Accepted Manuscript Development of direct conversion method for microalgal biodiesel production using wet biomass of Nannochloropsis salina Tae-Hyoung Kim, William I. Suh, Gursong Yoo, Sanjiv K. Mishra, Wasif Farooq, Myounghoon Moon, Anupama Shrivastav, Min S. Park, Ji-Won Yang PII: DOI: Reference:
S0960-8524(15)00363-6 http://dx.doi.org/10.1016/j.biortech.2015.03.033 BITE 14723
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
28 January 2015 4 March 2015 6 March 2015
Please cite this article as: Kim, T-H., Suh, W.I., Yoo, G., Mishra, S.K., Farooq, W., Moon, M., Shrivastav, A., Park, M.S., Yang, J-W., Development of direct conversion method for microalgal biodiesel production using wet biomass of Nannochloropsis salina, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.03.033
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Development of direct conversion method for microalgal biodiesel production using wet
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biomass of Nannochloropsis salina
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Tae-Hyoung Kim1, William I. Suh2, Gursong Yoo1, Sanjiv K. Mishra2, Wasif Farooq1,
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Myounghoon Moon1, Anupama Shrivastav2, Min S. Park1, 2, Ji-Won Yang1, 2*
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of Korea
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*Corresponding author, e-mail address: [email protected]
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TEL: +82-42-350-3964, FAX: +82-42-350-3910
Department of Chemical & Biomolecular Engineering, KAIST, Daejeon, 305-701, Republic
Advanced Biomass R&D Center, KAIST, Daejeon, 305-701, Republic of Korea
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Abstract
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In this work, the effects of several factors, such as temperature, reaction time, and
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solvent and acid quantity on in situ transesterification yield of wet Nannochloropsis salina
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were investigated. Under equivalent total solvent volume to biomass ratio, pure alcohol
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showed higher yield compared to alcohol-chloroform solvent. For esterifying 200 mg of wet
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cells, 2 ml of methanol and 1 ml of ethanol was sufficient to complete in situ
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transesterification. At 105 ºC or higher, 2.5% and 5% concentrations of sulfuric acid
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successfully converted more than 90% of lipid in 30 min. Also, it was verified that the
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optimal condition found in small-scale experiments is applicable to larger scale using 2 L
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scale reactor as well.
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Keywords:
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Nannochloropsis salina
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Microalgae;
Biodiesel;
Fatty
acid
alkyl
ester;
trans-esterification;
Introduction
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Concerns regarding depletion of easily accessible conventional petroleum reserves have
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lead to increased interests in alternative sources for transportation fuel. Even though shale gas
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has been recently getting widespread attention as future energy sources due to its wide
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availability, it is neither renewable nor carbon neutral (Shirvani et al., 2011). Biofuel is one
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possible solution which is eco-friendly and sustainable. Microalgae are one of the most
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promising precursors for biofuel, as microalgae can accumulate high level of lipids which can
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be converted into biodiesel. Microalgal biodiesel has several notable benefits compare to
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biodiesel from crop oil or bioethanol from grains or cellulosic biomass. Microalgae can
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produce much greater quantity of biomass compared to land plants per land area and time. In
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terms of biodiesel productivity, microalgae can yield 10 times greater productivity than 2
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jatropha and 50 times that of soybean (Amaro et al., 2011; Chisti, 2007; Halim et al., 2012;
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Mata et al., 2010; Scott et al., 2010). Moreover, microalgae can utilize waste water and
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nutrient resources that do not compete with human food production (Mata et al., 2010).
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However, a number of technical and economical hurdles must be overcome in order to
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achieve successful industrialization and commercialization of microalgal biodiesel (Halim
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et al., 2012). Lipid extraction and conversion steps are the major obstacles for the
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commercialization of microalgal biodiesel because of the high cost and energy input required.
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Extraction step takes more than 50% of total energy consumption even though drying step
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was omitted, which generally takes about 80% of total energy consumption. Ignoring
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cultivation, downstream steps takes 60% total production cost (Kim et al., 2013; Lardon et al.,
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2009; Wahidin et al., 2014).
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Among various lipid extraction/conversion methods for bio-feedstock including
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microalgae and crops, in situ transesterification (also called direct conversion or direct
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transesterification) is one of the most promising process for producing biodiesel in a fatty
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acid alkyl ester (FAAE) form (Griffiths et al., 2010). FAAE can be obtained by
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transesterification of triacylglyceride (TAG) or esterification of free fatty acid (FFA) with
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short chain alcohol. Methanol and ethanol are the most frequently used for transesterification,
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and the reaction results in the production of fatty acid methyl ester (FAME) or fatty acid ethyl
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ester (FAEE) respectively (Park et al., 2014a; Yusoff et al., 2014). Traditionally, extraction
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and conversion steps were independent and separate processes: lipid is first extracted from
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microalgal cells via one process, and undergoes transesterification in the second process.
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However, in situ transesterification can produce biodiesel with a single reactor using
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microalgae cells, not extracted lipid, as feed material. Therefore, this can be significantly 3
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reduce the energy consumption (Kim et al., 2013; Park et al., 2014c). In situ
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transesterification is also known to show higher productivity compared to most
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extraction/conversion processes as well (Cavonius et al., 2014; Griffiths et al., 2010). Some
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past research have studied in situ transesterification of microalgal cells. One study
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transesterified various strains of dried algal cells under different conditions to examine how
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much FAME can be produced (Wahlen et al., 2011). Another group compared the two step
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extraction/conversion methods and in situ transesterification with Schizochytrium limacinum
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by using various co-solvents (Johnson & Wen, 2009). One study using Nannocholopsis
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biomass attempted to incorporate an extra treatment using microwave and ultrasound
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radiation to improve the performance (Koberg et al., 2011).
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However, traditional in situ transesterification also has several disadvantages, with a
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major one being that an excessive amount of solvent and catalyst are required (Wahlen et al.,
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2011). In normal transesterification reaction, 3 alcohol molecules are theoretically needed to
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transesterify one TAG molecule, but in practicality 6 alcohol molecules are required for direct
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reaction between alcohol and pure oil (Fukuda et al., 2001). However, for in situ
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transesterification case, greater than 100:1 mol ratio is typically used in previous studies,
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since alcohol serves as not only just the reactant but the solvent for lipid extraction as well
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(Velasquez-Orta et al., 2012). In this process, lipid is extracted by alcohol and undergoes
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esterification reaction simultaneously. Therefore, excessive alcohol requirement is due to the
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lipid extraction part of the process. Moreover, cells include not only lipid but many other
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impurities that can hinder the reaction, such as cell debris and chlorophyll (Park et al., 2014b).
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Therefore, excessive reactant is needed to drive the reaction toward biodiesel production for
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the in situ transesterification of microalgae. 4
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Another disadvantage commonly present in most dry biomass based oil extraction
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processes, is a requirement of energy intensive cell drying step after harvesting the biomass
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(Kim et al., 2013). Drying steps are often performed before most downstream processes in
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order to achieve more effective extraction, faster reaction rate and higher conversion yield
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(Johnson & Wen, 2009; Kim et al., 2013). However, it is questionable if the drying step can
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improve the overall process economy due to the additional energy and cost incurred (Canakci
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& Van Gerpen, 1999). For that reason a number of studies evaluated downstream processes
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that can be performed using wet biomass. Some examples include transesterifying the cells in
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supercritical conditions (Levine et al., 2010; Patil et al., 2011), or using microwave to disrupt
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the cell wall prior to in situ transesterification (Cheng et al., 2013). However, these methods
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still require additional costs and high energy, which defeats the original purpose of using wet
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biomass. Therefore, transesterification of wet biomass without additional treatment should be
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explored in order to achieve improved economical outlook.
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There has been a number of previous research regarding the optimization of the in situ
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transesterification of microalgal biomass, but most of these used dry cells as a feedstock. In
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this study, the optimization of in situ transesterification of Nannochloropsis salina was
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investigated using wet biomass of the algae. Development of the process that uses wet algal
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biomass is particularly important, as life-cycle assessment of microalgal biodiesel reported
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that wet microalgae route greatly outperforms dry route in energy balance (Lardon et al.,
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2009). Since the goal of this study is to minimize the cost of the entire process, no additional
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step or method, such as supercritical condition or microwaves were used. The study
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optimized for the lowest reaction time, temperature, and amount of solvent and catalyst. The
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effects of solvent and moisture of cells were also studied. The optimized reaction condition 5
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was scaled up from 2 ml to 1 L scale in order to verify whether the optimized conditions are
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applicable on larger scale as well. The findings in this work are expected to greatly aid the
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development of successful algae biodiesel production process.
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2
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Materials and methods 2.1 Materials
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Nannochloropsis salina biomass cultivated in 200 ton raceway ponds was provided by
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NLP, a South Korean microalgae cultivation company. The microalgal sample was harvested
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by continuous centrifugation and frozen at -70 °C for long-term storage. It was thawed right
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before the series of experiments. HPLC-grade methanol, ethanol, and chloroform were
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purchased from Merck. Sulfuric acid (98%) and eicosane (≥99.5%) were purchased from
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Sigma-Aldrich, and used as homogenous catalyst for transesterification and standard material
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for quantitative analysis, respectively.
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2.2 In situ transesterification in small scale
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In every experiment, a frozen cell sample was thawed and washed by distilled water to
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discard bacteria and other nutrients from the culture. After washing, the sample was
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centrifuged at 7000 rpm for 5 min to discard the extracellular water. Then, 200 mg of wet
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cells were sampled into 14 ml Teflon-sealed glass tubes from Pyrex. Cell moisture (water
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content of the sample) was determined by the mass difference of freeze dried cell and wet cell
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sample, and its value was 76.5±1.0%.
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Alcohol and sulfuric acid were added to each biomass sample and were mixed. The
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amount of the reactant (alcohol) and catalyst (sulfuric acid) were varied from each
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experiment. After the addition, the tubes were capped very tightly and uniformly mixed for 5 6
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min by a vortexer (IKA vortex 3). The reaction was conducted in a heating block (DAIHAN
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Wisetherm HB-96D). When the reaction time was over, the tubes were cooled by cold water
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immediately.
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2.3 In situ transesterification in 2 L scale reactor
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To verify that the conditions from the series of previous small scale experiments is valid
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for larger scale as well, the experiment was scaled up to 2 liter batch reactor. The scale was
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designed to be approximately 500 times larger than the small scale optimization described in
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Section 2.2. Approximately 100 g of microalgal slurry was mixed with methanol or ethanol.
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After the mixture was homogenized, sulfuric acid was added to each sample. The solution
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were poured in the reactor and heated to a predetermined temperature. The solution was
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agitated by an impeller at 300 rpm. The reactor was cooled to ambient temperature after the
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reaction was run for a predetermined duration.
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2.4 Gas chromatography (GC) analysis
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After cooling, 2 ml of chloroform containing 0.5 mg of eicosane was added to reaction
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product sample for quantitative analysis. For phase separation, distilled water was added to
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each tube and centrifuged at 4000 rpm for 5 min. Then, chloroform, FAAE, and algal residues
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formed a lower phase, and water and alcohol formed an upper phase. The lower phase was
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moved to each 2 ml vial for GC after syringe filtration. The sample was analyzed by an
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Agilent 6890 GC with a HP-INNOWAX column (30 mm × 0.32 mm ×0.5 µm, Agilent, USA)
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and an FID detector.
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2.5 Determination of esterifiable lipid in cells
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The total esterifiable lipid was determined by in situ transesterification of dry cells at an
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excessive acid catalyst condition. A freeze dried cells 10 mg sample were vortexed for lipid 7
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extraction in 2 ml of chloroform-alcohol solution (2:1 v/v), and in situ transesterified for 30
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min at 100 °C after addition of 1 ml methanol and 300 µl sulfuric acid. The esterifiable lipid
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was expressed by the formula:
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Their values were 185.9±17.7 mg FAME/mg cell, and 188.4±21.9 mg FAEE/mg cell,
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respectively. The biodiesel yield for each experiment was calculated by biodiesel
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yield=
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3
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mg FAAE/mg cell.
100(%)
Results and discussion 3.1 Comparison between pure alcohol and alcohol-chloroform as solvent
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According to previous research, using co-solvent with alcohol can improve the
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performance of in situ transesterification, due to the fact that TAG has low solubility in pure
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alcohols. In particular, several studies report that chloroform has the highest performances
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among the various co-solvents due to the fact that it is highly miscible with both alcohols and
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lipids (Im et al., 2014; Johnson & Wen, 2009). To verify whether chloroform can positively
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influence in situ esterification using wet microalgal biomass, the performance of pure alcohol
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was compared with various ratios of alcohol-chloroform mixture. The ratio of alcohol-
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chloroform mixtures tested were of 2:1, 1:1 and 1:2 in volume (Folch et al., 1957; Im et al.,
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2014; Park et al., 2014c). The reaction was carried out with 2 ml of the solvent at 100 °C for
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1 h. (Figure 1). Contrary to other studies, pure alcohol showed better performance in both 50
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µl and 100 µl sulfuric acid conditions. The yield of the alcohol-chloroform solvent decreased
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as the alcohol fraction in the solvent was lowered. The overall ratio between the alcohol and
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the biomass decreases as the ratio between the alcohol and chloroform decreases. The
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combination of decreased solvent contact and lower concentration of alcohol, is thought to 8
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result in the decreased reaction yield observed when chloroform was added to the system. It
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was found that both pure methanol and ethanol achieved 100% FAAE yield at given
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condition.
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There are two factors determine the yield, namely extraction and conversion
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performances. Extraction efficiency is determined by the characteristics of the solvent such as
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solvent-lipid miscibility, which is primarily based on the polarity of the solvent (Johnson &
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Lusas, 1983). Conversion efficiency is determined by the concentration of reactant and
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catalyst (Canakci & Van Gerpen, 1999; Wahlen et al., 2008). It is widely reported that
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methanol-chloroform solution is an effective solvent system for lipid extraction
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(Balasubramanian et al., 2013). On the other hand, high polarity of pure methanol renders it
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immiscible with lipids and thus unsuitable for lipid extraction. However, in direct
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esterification, it can be argued that the complete solubilization of lipid within the solvent is
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not necessary. This is because the reactions take place within the intracellular compartments
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of the cells, between the lipid droplets and the alcohol that penetrated the cells. The lipids are
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contained within the algal cells as microscopic droplets, which provide plenty of surfaces
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area in which esterification can occur in situ, without necessitating complete solubilization. In
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addition, the resulting product of the reaction (FAAE) is relatively soluble in alcohols, which
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means the product can readily dissolve out into the solvent phase as the reaction proceeds.
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There are further benefits of not using chloroform as a co-solvent. Using chloroform in
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industry has been discouraged because it is highly toxic to the human health and environment.
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Not using co-solvent can also make the process simpler because the separation of co-solvent
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will not be needed. Therefore, we concluded that using chloroform as a co-solvent may not
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be a good choice for in situ transesterification of wet microalgal cells. Similar research which
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used methanol-chloroform solution as the solvent was compared to this study in Table 1. 9
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Despite milder reaction conditions, such as lower temperature, shorter reaction time, and less
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acid, the yield is much higher when pure methanol was used.
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3.2 Ratio of the solvent and biomass
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In general, as alcohol to biomass ratio increases, the FAAE yield from in situ
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esterification also also increases (Wahlen et al., 2011). In this experiment, different volume of
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alcohol was used for reaction with 200 mg of wet biomass in order to find optimal ratio of
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biomass and solvent. The total sulfuric acid quantity was fixed at 25 µl and 50 µl. The
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reaction temperature and time were fixed at 100 °C and 1 h, respectively. Under 25 µl of
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sulfuric acid, the highest yields were shown at 2 ml of methanol (88.1%) and 1 ml of ethanol
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(76.3%), respectively. In 50 µl sulfuric acid condition, the FAME yield reached 100% at 1.5
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ml, 2 ml and 2.5 ml of methanol. Every FAEE yield was over 90% except at 0.5 ml of ethanol.
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From these results, it was concluded that 2 ml of methanol and 1 ml of ethanol would be
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proper amount for in situ transesterification of 200 mg of wet microalgae.
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Though methanol requires milder reaction condition than ethanol (Yusoff et al., 2014), the
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peak FAEE yield was found at lower amounts of alcohol than that of FAME. In particular,
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when using 25 µl acid the FAEE yield peaked at 1 ml ethanol and decreased gradually with
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increasing ethanol volume, while the FAME yield paked at 2 ml methanol. The reason for
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decreasing of the yield, despite the increase of reactant, is most likely attributed to the lower
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concentration of sulfuric acid within the reaction, when the alcohol volume was increased.
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Meanwhile, the result that ethanol achieved complete FAAE yield at lower solvent volumes
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compared to that of methanol is likely explained the fact that neutral lipids have greater
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solubility under ethanol compared to methanol. According to previous research, methanol
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was able to only extract 3.1 mg of TAG from 100 mg of cells, while ethanol was able to 10
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extract 20.2 mg of TAG under the same condition (Wahlen et al., 2011). Therefore, it can be
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concluded that smaller amount of ethanol can esterify lipid as much as methanol with the
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same amount of sulfuric acid. However, due to the higher cost and lower reactivity of ethanol
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compared to methanol, it cannot be said that using ethanol is more economical for the in situ
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transesterification of microalgae.
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3.3 Effects of temperature and catalyst (sulfuric acid) concentration
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As the previous experiment concerning the solvent volume showed that the catalyst
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concentration substantially affects the the FAAE yields, this new series of experiments sought
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to find the proper concentration of catalyst under various temperatures and fixed solvent to
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biomass ratio. The reaction temperatures tested ranged from 75 °C to 120 °C for 1 h. For the
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catalyst concentration, 15 µl, 25 µl or 35 µl of sulfuric acid was added into each sample. The
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solvent volume was adjusted as 2 ml for methanol and 1 ml for ethanol, as the previous
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experiments showed that reaction with ethanol requires less solvent volume and higher acid
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concentration.
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The yield increased with temperature and acid concentration in every experiment (Figure
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3). Reaction with methanol resulted in a maximum of 98.4% yield under the highest
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temperature conditions. On the other hand, ethanol was able to achieve complete reaction
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under much milder conditions than methanol at given conditions despite having only 1 ml
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reaction volume, most likely due to the higher acid concentration. Both of the graphs show
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that the yield increased abruptly as temperature is raised from 75 °C to 90 °C, and after this
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area, FAAE yield increases more gradually. Therefore, it appears that the optimal target
238
temperature for lipid conversion step should be at least 90 °C at given acid concentration and
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reaction time. Because FAME was not sufficiently generated in 1 h, it was concluded that 11
240
more harsh condition, such as higher temperature, longer reaction time, or sulfuric acid
241
concentration higher than 1.75% (v/v) is needed for efficient FAME conversion.
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3.4 Effects of time and temperature on yield
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Reducing reaction time also can improve the overall process economy, as it does not only
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increase the amount of product generated per same unit time, but also reduces the energy
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required for maintaining the temperature. This experiment was designed to find the minimum
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temperature and reaction time for a complete reaction. In accordance to the result from
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Section 3.2, 2 ml of methanol or 1 ml of ethanol was used. The reaction temperatures tested
248
ranged from 75 °C to 120 °C, and the reaction time was varied from 30 min to 90 min.
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Sulfuric acid volume was fixed at 50 µl, and the alcohol volume was fixed at 2 ml and 1 ml
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for methanol and ethanol respectively (Figure 4).
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Similar to the result of Section 3.3, the temperature was found to greatly affect the FAAE
252
yields. In the methanol’s case, the largest increase in the yield was observed between 90 °C
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and 105 °C. After 30 min, more than 90% of FFA and TAG was esterified at 105 °C, while
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only 60% of FAME was generated at 90 °C. In ethanol’s case, similar jump in the yields was
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shown between 75 °C and 90 °C. At 5% acid concentration, FAEE yield remained low even
256
after 90 min at 75 °C, while almost complete reaction was shown after only 45 min of
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reaction at 90 °C. From these two experiments, it was concluded that the reaction time for the
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complete conversion can be reduced to less than 1 h, if the acid concentration and
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temperature are high enough. If higher concentration of acid can significantly lower the
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proper temperature and reaction time for complete conversion, it will be very helpful for the
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total energy balance of the whole process, as the cost of the additional acid is relatively
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smaller compared to the energy consumed in the overall operation. 12
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3.5 Effect of water content of the feed biomass
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To examine the effect of water content in the feed biomass on the reaction yield, the
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water content of the sample biomass was artificially adjusted via addition of distilled water to
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the compact wet biomass containing 75% moisture content. The moisture content was
267
adjusted to approximately 80% and 90%. The reaction temperature was set to 100 °C, 50 µl
268
of sulfuric acid, and 2 ml of methanol or ethanol was used.
269
The result is shown in Figure 5. Both of the samples with 75% and 80% water content
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was able to achieve maximum reaction yield. However, the rate of the reactions were
271
substantially different. Even merely 5% increase in moisture content nearly doubled the
272
reaction time required to reach 100% yield. In the case of 90% moisture content, it appeared
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that the reaction was incomplete even after 90 min of reaction at the given condition.
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Therefore, it is very important to decrease the water content in the cells at harvesting step in
275
order to minimize the reaction time and cost. It seems that compared to ethanol, methanol is
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more substantially effected by the moisture content of the cells. This can also be explained by
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ethanol’s lower polarity, which allows it to more effectively extract lipids even under slightly
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higher moisture content. Even though water also hinders the ability of ethanol to esterify the
279
lipid, ethanol appears to be able to better tolerate the presence of water in the sample.
280
However, its reactivity is still lower than that of methanol, as it took twice the duration to
281
reach 90% conversion yield at 75% moisture.
282
3.6 Scale-up experiment
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Because the scale was designed to be approximately 500 times larger than the small scale
284
experiments, 1 L of methanol or 500 ml of ethanol was used for each experiment. Detailed
285
condition and the corresponding small scale experiment is described in Table 2. At the given 13
286
condition, the yield from both of the experiments reached 100%, which means that the
287
optimized conditions found in the small scale experiments are applicable at larger scale as
288
well. Generally, the reaction performance becomes worse if the experimental scale is
289
enlarged, because of poor mass transfer and heat transfer. Therefore, this result indicates that
290
these conditions are enough to transesterify all the lipid as much as possible.
291
4
Conclusion
292
The optimal conditions for the in situ transesterification of wet Nannochloropsis salina
293
in lab scale were found by adjusting solvent, acid concentration, temperature, time, and
294
moisture. It was found that 10 ml of methanol or 5 ml of ethanol is proper for 1 g of wet
295
biomass, without co-solvent. Under 1 h reaction at 100 °C, 2.5% and 5% of sulfuric acid
296
(v/v) seems sufficient for complete conversion in methanol and ethanol, respectively. These
297
conditions were applicable at 0.5-1 L solvent systems as well. These data are expected to
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improve the overall economics of microalgal biodiesel production.
299
Acknowledgment
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This work was supported by the Advanced Biomass R&D Center (ABC) as the Global
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Frontier Project funded by the Ministry of Science, ICT, and Future Planning (ABC-2010-
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0029728). We also thank to NLP kindly provided the biomass for this study.
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339
in Situ Lipid Hydrolysis and Supercritical Transesterification. Energy & Fuels, 24(9), 5235-
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5243.
341 342 343 344 345 346
Mata, T.M., Martins, A.A., Caetano, N.S. 2010. Microalgae for biodiesel production and other applications: A review. Renew Sustain Energy Rev, 14(1), 217-232. Park, J.Y., Nam, B., Choi, S.A., Oh, Y.K., Lee, J.S. 2014a. Effects of anionic surfactant on extraction of free fatty acid from Chlorella vulgaris. Bioresour Technol, 166, 620-4. Park, J.Y., Oh, Y.K., Lee, J.S., Lee, K., Jeong, M.J., Choi, S.A. 2014b. Acid-catalyzed hot-water extraction of lipids from Chlorella vulgaris. Bioresour Technol, 153, 408-12. 15
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Park, J.Y., Park, M.S., Lee, Y.C., Yang, J.W. 2014c. Advances in direct transesterification of algal oils from wet biomass. Bioresour Technol.
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Patil, P.D., Gude, V.G., Mannarswamy, A., Deng, S., Cooke, P., Munson-McGee, S., Rhodes, I.,
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Lammers, P., Nirmalakhandan, N. 2011. Optimization of direct conversion of wet algae to
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biodiesel under supercritical methanol conditions. Bioresour Technol, 102(1), 118-22.
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Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G. 2010. Biodiesel
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from algae: challenges and prospects. Curr Opin Biotechnol, 21(3), 277-86. Shirvani, T., Yan, X., Inderwildi, O., Edwards, P., King, D. 2011. Life cycle energy and greenhouse gas analysis for algae-derived biodiesel. Energy & Environmental Science, 4, 3773-8. Velasquez-Orta, S.B., Lee, J.G.M., Harvey, A. 2012. Alkaline in situ transesterification of Chlorella vulgaris. Fuel, 94, 544-550. Wahidin, S., Idris, A., Shaleh, S.R.M. 2014. Rapid biodiesel production using wet microalgae via microwave irradiation. Energy Conversion and Management, 84, 227-233.
360
Wahlen, B.D., Barney, B.M., Seefeldt, L.C. 2008. Synthesis of Biodiesel from Mixed Feedstocks and
361
Longer Chain Alcohols Using an Acid-Catalyzed Method. Energy & Fuels, 22(6), 4223-4228.
362
Wahlen, B.D., Willis, R.M., Seefeldt, L.C. 2011. Biodiesel production by simultaneous extraction and
363
conversion of total lipids from microalgae, cyanobacteria, and wild mixed-cultures.
364
Bioresour Technol, 102(3), 2724-30.
365
Yusoff, M.F.M., Xu, X., Guo, Z. 2014. Comparison of Fatty Acid Methyl and Ethyl Esters as Biodiesel
366
Base Stock: a Review on Processing and Production Requirements. J Am Oil Chem Soc,
367
91(4), 525-531.
368 369
16
370 371 372
Figure Legends
373
Figure 1. Comparison of the yield in pure alcohol and alcohol-chloroform mixture. The
374
solvent were A) methanol and B) ethanol, respectively. Reaction temperature was 100 °C, and
375
2 ml of alcohol was used.
376
Figure 2. Comparison of various amount of alcohol in A) 25 µl and B) 50 µl.
377
Figure 3. A) FAME yield and B) FAEE yield with various amounts of sulfuric acid at each
378
temperature.
379
Figure 4. A) FAME yield and B) FAEE yield at various reaction time and temperature.
380
Figure 5. Comparison of A) FAME and B) FAEE yield with various cell moisture and
381
reaction time
382 383 384
17
100
A)
100
80
FAEE Yield (%)
FAME Yield (%)
80
B)
60
40
20
60
40
20
0
0 50 Alcohol : Chloroform (v:v)
Pure methanol 2:1 1:1 1:2
100
Sulfuric acid (l)
50 Alcohol : Chloroform (v:v)
100
Sulfuric acid (l)
Pure ethanol 2:1 1:1 1:2
Figure 1. Comparison of the yield in pure alcohol and alcohol-chloroform mixture. The solvent were A) methanol and B) ethanol, respectively. Reaction temperature was 100 °C, and 2 ml of alcohol was used.
A)
100
90
Yield (%)
90
Yield (%)
B)
100
80
70
80
70
60
60
Methanol Ethanol
50
Methanol Ethanol
50 0.0
0.5
1.0
1.5
Alcohol (ml)
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Alcohol (ml)
Figure 2. Comparison of FAAE yield in various amounts of alcohol in A) 25 μl and B) 50 μl.
110
110
B)
100
100
90
90
FAEE yield (%)
FAME yield (%)
A)
80 70 60 50
Sulfuric acid 35 l Sulfuric acid 25 l Sulfuric acid 15 l
40
80 70 60 50
Sulfuric acid 35 l Sulfuric acid 25 l Sulfuric acid 15 l
40 30
30 75
90
105
120
75
90
105
120
o
o
Temperature ( C)
Temperature ( C)
Figure 3. A) FAME yield and B) FAEE yield with various amounts of sulfuric acid at each temperature.
110
110
B)
100
100
90
90
80 70 120 ℃ 105 ℃ 90 ℃ 75 ℃
60 50 40
FAEE yield (%)
FAME yield (%)
A)
80 70 120 ℃ 105 ℃ 90 ℃ 75 ℃
60 50 40
30
45
60
Time (min)
75
90
30
45
60
75
90
Time (min)
Figure 4. A) FAME yield and B) FAEE yield at various reaction time and temperature.
100
A)
100 90
FAEE yield (%)
FAME yield (%)
90
B)
80 70 60 50
76.36% 81.85% 90.80%
40 15
30
45
60
75
80 70 60 50
74.87% 81.45% 90.36%
40
90
Time (min)
15
30
45
60
75
90
Time (min)
Figure 5. Comparison of A) FAME and B) FAEE yield with various cell moisture and reaction time
411 412
Table 1. Comparison to other research
This work
(Im et al., 2014)
Species
Nannochloropsis salina
Nannochloropsis oceanica
Esterifiable lipid (dry)
185.9±17.7 mg FAME/g cell
191.7±8.2 mg FAME/g cell
Moisture
76.5±1.0%
65 % MeOH 1 ml
MeOH 1 ml
CHCl3 2 ml
CHCl3 2 ml
105 °C
95 °C
95 °C
50 µl
50 µl
300 µl
100 µl
60 min
60 min
30 min
60 min
120 min
94.2 %
99.8 %
91.5 %
84.7 %
82.8 %
Solvent
MeOH 2 ml
MeOH 1.5 ml
MeOH 2 ml
Temperature
90 °C
100 °C
Sulfuric acid
35 µl
Time Yield 413
414
23
415 416
417
Table 2. Comparison of small scale and large scale experiments
Small scale
Large scale
Biomass
200 mg (wet)
100 g (wet)
Solvent
MeOH 2 ml
MeOH 1 L
Catalyst
H2SO4 50 µl
H2SO4 30 ml
Temperature
100 °C
100 °C
Time
1h
1h
FAME Yield
99.7 %
100.4 %
Small scale
Large scale
Biomass
200 mg (wet)
100 g (wet)
Solvent
EtOH 1 ml
EtOH 0.5 L
Catalyst
H2SO4 50 µl
H2SO4 30 ml
Temperature
100 °C
100 °C
Time
1h
1h
FAEE Yield
94.6 %
101.0 %
418
419
420 421 422 423
24
424
Highlights
425
•
FAME and FAEE can be produced from the wet biomass in situ.
426
•
Acid catalyst was used for direct transesterification of wet biomass to generate
427
FAAEs
428
•
High yield (>90% of esterifiable lipids) was achieved.
429
•
At > 105 ºC, 2.5- 5% of H2SO4 successfully converted more than 90% of lipids.
430
25
S0960-8524(15)00363-6 http://dx.doi.org/10.1016/j.biortech.2015.03.033 BITE 14723
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
28 January 2015 4 March 2015 6 March 2015
Please cite this article as: Kim, T-H., Suh, W.I., Yoo, G., Mishra, S.K., Farooq, W., Moon, M., Shrivastav, A., Park, M.S., Yang, J-W., Development of direct conversion method for microalgal biodiesel production using wet biomass of Nannochloropsis salina, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.03.033
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.
1
Development of direct conversion method for microalgal biodiesel production using wet
2
biomass of Nannochloropsis salina
3
Tae-Hyoung Kim1, William I. Suh2, Gursong Yoo1, Sanjiv K. Mishra2, Wasif Farooq1,
4
Myounghoon Moon1, Anupama Shrivastav2, Min S. Park1, 2, Ji-Won Yang1, 2*
5
1
6
of Korea
7
2
8
*Corresponding author, e-mail address: [email protected]
9
TEL: +82-42-350-3964, FAX: +82-42-350-3910
Department of Chemical & Biomolecular Engineering, KAIST, Daejeon, 305-701, Republic
Advanced Biomass R&D Center, KAIST, Daejeon, 305-701, Republic of Korea
10
1
11
Abstract
12
In this work, the effects of several factors, such as temperature, reaction time, and
13
solvent and acid quantity on in situ transesterification yield of wet Nannochloropsis salina
14
were investigated. Under equivalent total solvent volume to biomass ratio, pure alcohol
15
showed higher yield compared to alcohol-chloroform solvent. For esterifying 200 mg of wet
16
cells, 2 ml of methanol and 1 ml of ethanol was sufficient to complete in situ
17
transesterification. At 105 ºC or higher, 2.5% and 5% concentrations of sulfuric acid
18
successfully converted more than 90% of lipid in 30 min. Also, it was verified that the
19
optimal condition found in small-scale experiments is applicable to larger scale using 2 L
20
scale reactor as well.
21
Keywords:
22
Nannochloropsis salina
23
1
Microalgae;
Biodiesel;
Fatty
acid
alkyl
ester;
trans-esterification;
Introduction
24
Concerns regarding depletion of easily accessible conventional petroleum reserves have
25
lead to increased interests in alternative sources for transportation fuel. Even though shale gas
26
has been recently getting widespread attention as future energy sources due to its wide
27
availability, it is neither renewable nor carbon neutral (Shirvani et al., 2011). Biofuel is one
28
possible solution which is eco-friendly and sustainable. Microalgae are one of the most
29
promising precursors for biofuel, as microalgae can accumulate high level of lipids which can
30
be converted into biodiesel. Microalgal biodiesel has several notable benefits compare to
31
biodiesel from crop oil or bioethanol from grains or cellulosic biomass. Microalgae can
32
produce much greater quantity of biomass compared to land plants per land area and time. In
33
terms of biodiesel productivity, microalgae can yield 10 times greater productivity than 2
34
jatropha and 50 times that of soybean (Amaro et al., 2011; Chisti, 2007; Halim et al., 2012;
35
Mata et al., 2010; Scott et al., 2010). Moreover, microalgae can utilize waste water and
36
nutrient resources that do not compete with human food production (Mata et al., 2010).
37
However, a number of technical and economical hurdles must be overcome in order to
38
achieve successful industrialization and commercialization of microalgal biodiesel (Halim
39
et al., 2012). Lipid extraction and conversion steps are the major obstacles for the
40
commercialization of microalgal biodiesel because of the high cost and energy input required.
41
Extraction step takes more than 50% of total energy consumption even though drying step
42
was omitted, which generally takes about 80% of total energy consumption. Ignoring
43
cultivation, downstream steps takes 60% total production cost (Kim et al., 2013; Lardon et al.,
44
2009; Wahidin et al., 2014).
45
Among various lipid extraction/conversion methods for bio-feedstock including
46
microalgae and crops, in situ transesterification (also called direct conversion or direct
47
transesterification) is one of the most promising process for producing biodiesel in a fatty
48
acid alkyl ester (FAAE) form (Griffiths et al., 2010). FAAE can be obtained by
49
transesterification of triacylglyceride (TAG) or esterification of free fatty acid (FFA) with
50
short chain alcohol. Methanol and ethanol are the most frequently used for transesterification,
51
and the reaction results in the production of fatty acid methyl ester (FAME) or fatty acid ethyl
52
ester (FAEE) respectively (Park et al., 2014a; Yusoff et al., 2014). Traditionally, extraction
53
and conversion steps were independent and separate processes: lipid is first extracted from
54
microalgal cells via one process, and undergoes transesterification in the second process.
55
However, in situ transesterification can produce biodiesel with a single reactor using
56
microalgae cells, not extracted lipid, as feed material. Therefore, this can be significantly 3
57
reduce the energy consumption (Kim et al., 2013; Park et al., 2014c). In situ
58
transesterification is also known to show higher productivity compared to most
59
extraction/conversion processes as well (Cavonius et al., 2014; Griffiths et al., 2010). Some
60
past research have studied in situ transesterification of microalgal cells. One study
61
transesterified various strains of dried algal cells under different conditions to examine how
62
much FAME can be produced (Wahlen et al., 2011). Another group compared the two step
63
extraction/conversion methods and in situ transesterification with Schizochytrium limacinum
64
by using various co-solvents (Johnson & Wen, 2009). One study using Nannocholopsis
65
biomass attempted to incorporate an extra treatment using microwave and ultrasound
66
radiation to improve the performance (Koberg et al., 2011).
67
However, traditional in situ transesterification also has several disadvantages, with a
68
major one being that an excessive amount of solvent and catalyst are required (Wahlen et al.,
69
2011). In normal transesterification reaction, 3 alcohol molecules are theoretically needed to
70
transesterify one TAG molecule, but in practicality 6 alcohol molecules are required for direct
71
reaction between alcohol and pure oil (Fukuda et al., 2001). However, for in situ
72
transesterification case, greater than 100:1 mol ratio is typically used in previous studies,
73
since alcohol serves as not only just the reactant but the solvent for lipid extraction as well
74
(Velasquez-Orta et al., 2012). In this process, lipid is extracted by alcohol and undergoes
75
esterification reaction simultaneously. Therefore, excessive alcohol requirement is due to the
76
lipid extraction part of the process. Moreover, cells include not only lipid but many other
77
impurities that can hinder the reaction, such as cell debris and chlorophyll (Park et al., 2014b).
78
Therefore, excessive reactant is needed to drive the reaction toward biodiesel production for
79
the in situ transesterification of microalgae. 4
80
Another disadvantage commonly present in most dry biomass based oil extraction
81
processes, is a requirement of energy intensive cell drying step after harvesting the biomass
82
(Kim et al., 2013). Drying steps are often performed before most downstream processes in
83
order to achieve more effective extraction, faster reaction rate and higher conversion yield
84
(Johnson & Wen, 2009; Kim et al., 2013). However, it is questionable if the drying step can
85
improve the overall process economy due to the additional energy and cost incurred (Canakci
86
& Van Gerpen, 1999). For that reason a number of studies evaluated downstream processes
87
that can be performed using wet biomass. Some examples include transesterifying the cells in
88
supercritical conditions (Levine et al., 2010; Patil et al., 2011), or using microwave to disrupt
89
the cell wall prior to in situ transesterification (Cheng et al., 2013). However, these methods
90
still require additional costs and high energy, which defeats the original purpose of using wet
91
biomass. Therefore, transesterification of wet biomass without additional treatment should be
92
explored in order to achieve improved economical outlook.
93
There has been a number of previous research regarding the optimization of the in situ
94
transesterification of microalgal biomass, but most of these used dry cells as a feedstock. In
95
this study, the optimization of in situ transesterification of Nannochloropsis salina was
96
investigated using wet biomass of the algae. Development of the process that uses wet algal
97
biomass is particularly important, as life-cycle assessment of microalgal biodiesel reported
98
that wet microalgae route greatly outperforms dry route in energy balance (Lardon et al.,
99
2009). Since the goal of this study is to minimize the cost of the entire process, no additional
100
step or method, such as supercritical condition or microwaves were used. The study
101
optimized for the lowest reaction time, temperature, and amount of solvent and catalyst. The
102
effects of solvent and moisture of cells were also studied. The optimized reaction condition 5
103
was scaled up from 2 ml to 1 L scale in order to verify whether the optimized conditions are
104
applicable on larger scale as well. The findings in this work are expected to greatly aid the
105
development of successful algae biodiesel production process.
106
2
107
Materials and methods 2.1 Materials
108
Nannochloropsis salina biomass cultivated in 200 ton raceway ponds was provided by
109
NLP, a South Korean microalgae cultivation company. The microalgal sample was harvested
110
by continuous centrifugation and frozen at -70 °C for long-term storage. It was thawed right
111
before the series of experiments. HPLC-grade methanol, ethanol, and chloroform were
112
purchased from Merck. Sulfuric acid (98%) and eicosane (≥99.5%) were purchased from
113
Sigma-Aldrich, and used as homogenous catalyst for transesterification and standard material
114
for quantitative analysis, respectively.
115
2.2 In situ transesterification in small scale
116
In every experiment, a frozen cell sample was thawed and washed by distilled water to
117
discard bacteria and other nutrients from the culture. After washing, the sample was
118
centrifuged at 7000 rpm for 5 min to discard the extracellular water. Then, 200 mg of wet
119
cells were sampled into 14 ml Teflon-sealed glass tubes from Pyrex. Cell moisture (water
120
content of the sample) was determined by the mass difference of freeze dried cell and wet cell
121
sample, and its value was 76.5±1.0%.
122
Alcohol and sulfuric acid were added to each biomass sample and were mixed. The
123
amount of the reactant (alcohol) and catalyst (sulfuric acid) were varied from each
124
experiment. After the addition, the tubes were capped very tightly and uniformly mixed for 5 6
125
min by a vortexer (IKA vortex 3). The reaction was conducted in a heating block (DAIHAN
126
Wisetherm HB-96D). When the reaction time was over, the tubes were cooled by cold water
127
immediately.
128
2.3 In situ transesterification in 2 L scale reactor
129
To verify that the conditions from the series of previous small scale experiments is valid
130
for larger scale as well, the experiment was scaled up to 2 liter batch reactor. The scale was
131
designed to be approximately 500 times larger than the small scale optimization described in
132
Section 2.2. Approximately 100 g of microalgal slurry was mixed with methanol or ethanol.
133
After the mixture was homogenized, sulfuric acid was added to each sample. The solution
134
were poured in the reactor and heated to a predetermined temperature. The solution was
135
agitated by an impeller at 300 rpm. The reactor was cooled to ambient temperature after the
136
reaction was run for a predetermined duration.
137
2.4 Gas chromatography (GC) analysis
138
After cooling, 2 ml of chloroform containing 0.5 mg of eicosane was added to reaction
139
product sample for quantitative analysis. For phase separation, distilled water was added to
140
each tube and centrifuged at 4000 rpm for 5 min. Then, chloroform, FAAE, and algal residues
141
formed a lower phase, and water and alcohol formed an upper phase. The lower phase was
142
moved to each 2 ml vial for GC after syringe filtration. The sample was analyzed by an
143
Agilent 6890 GC with a HP-INNOWAX column (30 mm × 0.32 mm ×0.5 µm, Agilent, USA)
144
and an FID detector.
145
2.5 Determination of esterifiable lipid in cells
146
The total esterifiable lipid was determined by in situ transesterification of dry cells at an
147
excessive acid catalyst condition. A freeze dried cells 10 mg sample were vortexed for lipid 7
148
extraction in 2 ml of chloroform-alcohol solution (2:1 v/v), and in situ transesterified for 30
149
min at 100 °C after addition of 1 ml methanol and 300 µl sulfuric acid. The esterifiable lipid
150
was expressed by the formula:
151
Their values were 185.9±17.7 mg FAME/mg cell, and 188.4±21.9 mg FAEE/mg cell,
152
respectively. The biodiesel yield for each experiment was calculated by biodiesel
153
yield=
154
3
155
mg FAAE/mg cell.
100(%)
Results and discussion 3.1 Comparison between pure alcohol and alcohol-chloroform as solvent
156
According to previous research, using co-solvent with alcohol can improve the
157
performance of in situ transesterification, due to the fact that TAG has low solubility in pure
158
alcohols. In particular, several studies report that chloroform has the highest performances
159
among the various co-solvents due to the fact that it is highly miscible with both alcohols and
160
lipids (Im et al., 2014; Johnson & Wen, 2009). To verify whether chloroform can positively
161
influence in situ esterification using wet microalgal biomass, the performance of pure alcohol
162
was compared with various ratios of alcohol-chloroform mixture. The ratio of alcohol-
163
chloroform mixtures tested were of 2:1, 1:1 and 1:2 in volume (Folch et al., 1957; Im et al.,
164
2014; Park et al., 2014c). The reaction was carried out with 2 ml of the solvent at 100 °C for
165
1 h. (Figure 1). Contrary to other studies, pure alcohol showed better performance in both 50
166
µl and 100 µl sulfuric acid conditions. The yield of the alcohol-chloroform solvent decreased
167
as the alcohol fraction in the solvent was lowered. The overall ratio between the alcohol and
168
the biomass decreases as the ratio between the alcohol and chloroform decreases. The
169
combination of decreased solvent contact and lower concentration of alcohol, is thought to 8
170
result in the decreased reaction yield observed when chloroform was added to the system. It
171
was found that both pure methanol and ethanol achieved 100% FAAE yield at given
172
condition.
173
There are two factors determine the yield, namely extraction and conversion
174
performances. Extraction efficiency is determined by the characteristics of the solvent such as
175
solvent-lipid miscibility, which is primarily based on the polarity of the solvent (Johnson &
176
Lusas, 1983). Conversion efficiency is determined by the concentration of reactant and
177
catalyst (Canakci & Van Gerpen, 1999; Wahlen et al., 2008). It is widely reported that
178
methanol-chloroform solution is an effective solvent system for lipid extraction
179
(Balasubramanian et al., 2013). On the other hand, high polarity of pure methanol renders it
180
immiscible with lipids and thus unsuitable for lipid extraction. However, in direct
181
esterification, it can be argued that the complete solubilization of lipid within the solvent is
182
not necessary. This is because the reactions take place within the intracellular compartments
183
of the cells, between the lipid droplets and the alcohol that penetrated the cells. The lipids are
184
contained within the algal cells as microscopic droplets, which provide plenty of surfaces
185
area in which esterification can occur in situ, without necessitating complete solubilization. In
186
addition, the resulting product of the reaction (FAAE) is relatively soluble in alcohols, which
187
means the product can readily dissolve out into the solvent phase as the reaction proceeds.
188
There are further benefits of not using chloroform as a co-solvent. Using chloroform in
189
industry has been discouraged because it is highly toxic to the human health and environment.
190
Not using co-solvent can also make the process simpler because the separation of co-solvent
191
will not be needed. Therefore, we concluded that using chloroform as a co-solvent may not
192
be a good choice for in situ transesterification of wet microalgal cells. Similar research which
193
used methanol-chloroform solution as the solvent was compared to this study in Table 1. 9
194
Despite milder reaction conditions, such as lower temperature, shorter reaction time, and less
195
acid, the yield is much higher when pure methanol was used.
196
3.2 Ratio of the solvent and biomass
197
In general, as alcohol to biomass ratio increases, the FAAE yield from in situ
198
esterification also also increases (Wahlen et al., 2011). In this experiment, different volume of
199
alcohol was used for reaction with 200 mg of wet biomass in order to find optimal ratio of
200
biomass and solvent. The total sulfuric acid quantity was fixed at 25 µl and 50 µl. The
201
reaction temperature and time were fixed at 100 °C and 1 h, respectively. Under 25 µl of
202
sulfuric acid, the highest yields were shown at 2 ml of methanol (88.1%) and 1 ml of ethanol
203
(76.3%), respectively. In 50 µl sulfuric acid condition, the FAME yield reached 100% at 1.5
204
ml, 2 ml and 2.5 ml of methanol. Every FAEE yield was over 90% except at 0.5 ml of ethanol.
205
From these results, it was concluded that 2 ml of methanol and 1 ml of ethanol would be
206
proper amount for in situ transesterification of 200 mg of wet microalgae.
207
Though methanol requires milder reaction condition than ethanol (Yusoff et al., 2014), the
208
peak FAEE yield was found at lower amounts of alcohol than that of FAME. In particular,
209
when using 25 µl acid the FAEE yield peaked at 1 ml ethanol and decreased gradually with
210
increasing ethanol volume, while the FAME yield paked at 2 ml methanol. The reason for
211
decreasing of the yield, despite the increase of reactant, is most likely attributed to the lower
212
concentration of sulfuric acid within the reaction, when the alcohol volume was increased.
213
Meanwhile, the result that ethanol achieved complete FAAE yield at lower solvent volumes
214
compared to that of methanol is likely explained the fact that neutral lipids have greater
215
solubility under ethanol compared to methanol. According to previous research, methanol
216
was able to only extract 3.1 mg of TAG from 100 mg of cells, while ethanol was able to 10
217
extract 20.2 mg of TAG under the same condition (Wahlen et al., 2011). Therefore, it can be
218
concluded that smaller amount of ethanol can esterify lipid as much as methanol with the
219
same amount of sulfuric acid. However, due to the higher cost and lower reactivity of ethanol
220
compared to methanol, it cannot be said that using ethanol is more economical for the in situ
221
transesterification of microalgae.
222
3.3 Effects of temperature and catalyst (sulfuric acid) concentration
223
As the previous experiment concerning the solvent volume showed that the catalyst
224
concentration substantially affects the the FAAE yields, this new series of experiments sought
225
to find the proper concentration of catalyst under various temperatures and fixed solvent to
226
biomass ratio. The reaction temperatures tested ranged from 75 °C to 120 °C for 1 h. For the
227
catalyst concentration, 15 µl, 25 µl or 35 µl of sulfuric acid was added into each sample. The
228
solvent volume was adjusted as 2 ml for methanol and 1 ml for ethanol, as the previous
229
experiments showed that reaction with ethanol requires less solvent volume and higher acid
230
concentration.
231
The yield increased with temperature and acid concentration in every experiment (Figure
232
3). Reaction with methanol resulted in a maximum of 98.4% yield under the highest
233
temperature conditions. On the other hand, ethanol was able to achieve complete reaction
234
under much milder conditions than methanol at given conditions despite having only 1 ml
235
reaction volume, most likely due to the higher acid concentration. Both of the graphs show
236
that the yield increased abruptly as temperature is raised from 75 °C to 90 °C, and after this
237
area, FAAE yield increases more gradually. Therefore, it appears that the optimal target
238
temperature for lipid conversion step should be at least 90 °C at given acid concentration and
239
reaction time. Because FAME was not sufficiently generated in 1 h, it was concluded that 11
240
more harsh condition, such as higher temperature, longer reaction time, or sulfuric acid
241
concentration higher than 1.75% (v/v) is needed for efficient FAME conversion.
242
3.4 Effects of time and temperature on yield
243
Reducing reaction time also can improve the overall process economy, as it does not only
244
increase the amount of product generated per same unit time, but also reduces the energy
245
required for maintaining the temperature. This experiment was designed to find the minimum
246
temperature and reaction time for a complete reaction. In accordance to the result from
247
Section 3.2, 2 ml of methanol or 1 ml of ethanol was used. The reaction temperatures tested
248
ranged from 75 °C to 120 °C, and the reaction time was varied from 30 min to 90 min.
249
Sulfuric acid volume was fixed at 50 µl, and the alcohol volume was fixed at 2 ml and 1 ml
250
for methanol and ethanol respectively (Figure 4).
251
Similar to the result of Section 3.3, the temperature was found to greatly affect the FAAE
252
yields. In the methanol’s case, the largest increase in the yield was observed between 90 °C
253
and 105 °C. After 30 min, more than 90% of FFA and TAG was esterified at 105 °C, while
254
only 60% of FAME was generated at 90 °C. In ethanol’s case, similar jump in the yields was
255
shown between 75 °C and 90 °C. At 5% acid concentration, FAEE yield remained low even
256
after 90 min at 75 °C, while almost complete reaction was shown after only 45 min of
257
reaction at 90 °C. From these two experiments, it was concluded that the reaction time for the
258
complete conversion can be reduced to less than 1 h, if the acid concentration and
259
temperature are high enough. If higher concentration of acid can significantly lower the
260
proper temperature and reaction time for complete conversion, it will be very helpful for the
261
total energy balance of the whole process, as the cost of the additional acid is relatively
262
smaller compared to the energy consumed in the overall operation. 12
263
3.5 Effect of water content of the feed biomass
264
To examine the effect of water content in the feed biomass on the reaction yield, the
265
water content of the sample biomass was artificially adjusted via addition of distilled water to
266
the compact wet biomass containing 75% moisture content. The moisture content was
267
adjusted to approximately 80% and 90%. The reaction temperature was set to 100 °C, 50 µl
268
of sulfuric acid, and 2 ml of methanol or ethanol was used.
269
The result is shown in Figure 5. Both of the samples with 75% and 80% water content
270
was able to achieve maximum reaction yield. However, the rate of the reactions were
271
substantially different. Even merely 5% increase in moisture content nearly doubled the
272
reaction time required to reach 100% yield. In the case of 90% moisture content, it appeared
273
that the reaction was incomplete even after 90 min of reaction at the given condition.
274
Therefore, it is very important to decrease the water content in the cells at harvesting step in
275
order to minimize the reaction time and cost. It seems that compared to ethanol, methanol is
276
more substantially effected by the moisture content of the cells. This can also be explained by
277
ethanol’s lower polarity, which allows it to more effectively extract lipids even under slightly
278
higher moisture content. Even though water also hinders the ability of ethanol to esterify the
279
lipid, ethanol appears to be able to better tolerate the presence of water in the sample.
280
However, its reactivity is still lower than that of methanol, as it took twice the duration to
281
reach 90% conversion yield at 75% moisture.
282
3.6 Scale-up experiment
283
Because the scale was designed to be approximately 500 times larger than the small scale
284
experiments, 1 L of methanol or 500 ml of ethanol was used for each experiment. Detailed
285
condition and the corresponding small scale experiment is described in Table 2. At the given 13
286
condition, the yield from both of the experiments reached 100%, which means that the
287
optimized conditions found in the small scale experiments are applicable at larger scale as
288
well. Generally, the reaction performance becomes worse if the experimental scale is
289
enlarged, because of poor mass transfer and heat transfer. Therefore, this result indicates that
290
these conditions are enough to transesterify all the lipid as much as possible.
291
4
Conclusion
292
The optimal conditions for the in situ transesterification of wet Nannochloropsis salina
293
in lab scale were found by adjusting solvent, acid concentration, temperature, time, and
294
moisture. It was found that 10 ml of methanol or 5 ml of ethanol is proper for 1 g of wet
295
biomass, without co-solvent. Under 1 h reaction at 100 °C, 2.5% and 5% of sulfuric acid
296
(v/v) seems sufficient for complete conversion in methanol and ethanol, respectively. These
297
conditions were applicable at 0.5-1 L solvent systems as well. These data are expected to
298
improve the overall economics of microalgal biodiesel production.
299
Acknowledgment
300
This work was supported by the Advanced Biomass R&D Center (ABC) as the Global
301
Frontier Project funded by the Ministry of Science, ICT, and Future Planning (ABC-2010-
302
0029728). We also thank to NLP kindly provided the biomass for this study.
303
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Figure Legends
373
Figure 1. Comparison of the yield in pure alcohol and alcohol-chloroform mixture. The
374
solvent were A) methanol and B) ethanol, respectively. Reaction temperature was 100 °C, and
375
2 ml of alcohol was used.
376
Figure 2. Comparison of various amount of alcohol in A) 25 µl and B) 50 µl.
377
Figure 3. A) FAME yield and B) FAEE yield with various amounts of sulfuric acid at each
378
temperature.
379
Figure 4. A) FAME yield and B) FAEE yield at various reaction time and temperature.
380
Figure 5. Comparison of A) FAME and B) FAEE yield with various cell moisture and
381
reaction time
382 383 384
17
100
A)
100
80
FAEE Yield (%)
FAME Yield (%)
80
B)
60
40
20
60
40
20
0
0 50 Alcohol : Chloroform (v:v)
Pure methanol 2:1 1:1 1:2
100
Sulfuric acid (l)
50 Alcohol : Chloroform (v:v)
100
Sulfuric acid (l)
Pure ethanol 2:1 1:1 1:2
Figure 1. Comparison of the yield in pure alcohol and alcohol-chloroform mixture. The solvent were A) methanol and B) ethanol, respectively. Reaction temperature was 100 °C, and 2 ml of alcohol was used.
A)
100
90
Yield (%)
90
Yield (%)
B)
100
80
70
80
70
60
60
Methanol Ethanol
50
Methanol Ethanol
50 0.0
0.5
1.0
1.5
Alcohol (ml)
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Alcohol (ml)
Figure 2. Comparison of FAAE yield in various amounts of alcohol in A) 25 μl and B) 50 μl.
110
110
B)
100
100
90
90
FAEE yield (%)
FAME yield (%)
A)
80 70 60 50
Sulfuric acid 35 l Sulfuric acid 25 l Sulfuric acid 15 l
40
80 70 60 50
Sulfuric acid 35 l Sulfuric acid 25 l Sulfuric acid 15 l
40 30
30 75
90
105
120
75
90
105
120
o
o
Temperature ( C)
Temperature ( C)
Figure 3. A) FAME yield and B) FAEE yield with various amounts of sulfuric acid at each temperature.
110
110
B)
100
100
90
90
80 70 120 ℃ 105 ℃ 90 ℃ 75 ℃
60 50 40
FAEE yield (%)
FAME yield (%)
A)
80 70 120 ℃ 105 ℃ 90 ℃ 75 ℃
60 50 40
30
45
60
Time (min)
75
90
30
45
60
75
90
Time (min)
Figure 4. A) FAME yield and B) FAEE yield at various reaction time and temperature.
100
A)
100 90
FAEE yield (%)
FAME yield (%)
90
B)
80 70 60 50
76.36% 81.85% 90.80%
40 15
30
45
60
75
80 70 60 50
74.87% 81.45% 90.36%
40
90
Time (min)
15
30
45
60
75
90
Time (min)
Figure 5. Comparison of A) FAME and B) FAEE yield with various cell moisture and reaction time
411 412
Table 1. Comparison to other research
This work
(Im et al., 2014)
Species
Nannochloropsis salina
Nannochloropsis oceanica
Esterifiable lipid (dry)
185.9±17.7 mg FAME/g cell
191.7±8.2 mg FAME/g cell
Moisture
76.5±1.0%
65 % MeOH 1 ml
MeOH 1 ml
CHCl3 2 ml
CHCl3 2 ml
105 °C
95 °C
95 °C
50 µl
50 µl
300 µl
100 µl
60 min
60 min
30 min
60 min
120 min
94.2 %
99.8 %
91.5 %
84.7 %
82.8 %
Solvent
MeOH 2 ml
MeOH 1.5 ml
MeOH 2 ml
Temperature
90 °C
100 °C
Sulfuric acid
35 µl
Time Yield 413
414
23
415 416
417
Table 2. Comparison of small scale and large scale experiments
Small scale
Large scale
Biomass
200 mg (wet)
100 g (wet)
Solvent
MeOH 2 ml
MeOH 1 L
Catalyst
H2SO4 50 µl
H2SO4 30 ml
Temperature
100 °C
100 °C
Time
1h
1h
FAME Yield
99.7 %
100.4 %
Small scale
Large scale
Biomass
200 mg (wet)
100 g (wet)
Solvent
EtOH 1 ml
EtOH 0.5 L
Catalyst
H2SO4 50 µl
H2SO4 30 ml
Temperature
100 °C
100 °C
Time
1h
1h
FAEE Yield
94.6 %
101.0 %
418
419
420 421 422 423
24
424
Highlights
425
•
FAME and FAEE can be produced from the wet biomass in situ.
426
•
Acid catalyst was used for direct transesterification of wet biomass to generate
427
FAAEs
428
•
High yield (>90% of esterifiable lipids) was achieved.
429
•
At > 105 ºC, 2.5- 5% of H2SO4 successfully converted more than 90% of lipids.
430
25
