Beyond Fatty Acid Methyl Esters - American Chemical Society

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Beyond Fatty Acid Methyl Esters: Expanding the Renewable Carbon Profile with Alkenones from Isochrysis sp. Gregory W. O’Neil,*,† Catherine A. Carmichael,‡ Tyler J. Goepfert,‡,§ James M. Fulton,‡ Gerhard Knothe,∥ Connie Pui Ling Lau,⊥ Scott R. Lindell,# Nagwa G-E. Mohammady,⊥,¶ Benjamin A. S. Van Mooy,‡ and Christopher M. Reddy‡ †

Department of Chemistry, Western Washington University, Bellingham, Washington 98225, United States Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution (WHOI), Woods Hole, Massachusetts 02543, United States § Institute of Physics, PPRE Fak 5, Oldenburg Universität, 26111 Oldenburg, Germany ∥ National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois 61604, United States ⊥ Simon F. S. Li Marine Science Laboratory, The Chinese University of Hong Kong, Shaitin, New Territories, Hong Kong # Scientific Aquaculture Program, Marine Biological Laboratory (MBL), Woods Hole Oceanographic Institution (WHOI), Woods Hole, Massachusetts 02543, United States ¶ Department of Botany, Faculty of Science, Alexandria University, Alexandria 21526, Egypt ‡

S Supporting Information *

ABSTRACT: In addition to characteristic fatty acid methyl esters (FAMEs), biodiesel produced from Isochrysis sp. contains a significant amount (14%, w/w) of predominantly C37 and C38 long-chain alkenones. These compounds are members of a class of lipids known collectively as polyunsaturated long-chain alkenones (PULCAs) that are produced by a range of other prymnesiophyte taxa. The physical properties of alkenones, such as high melting points (∼60 °C), renders the direct product unsuitable for use as a diesel fuel but, nonetheless, represents an important and as yet unexplored renewable carbon feedstock.

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algal biomass1,16,17 and consider the impact of FAME contamination by the highly stable polyunsaturated long-chain alkenones present in Isochrysis. Long-chain (mainly C37 and C38) unsaturated methyl and ethyl ketones (alkenones) are part of a group of unusual compounds, including related alkenes and alkenoates, collectively referred to as polyunsaturated longchain alkenones (PULCAs) (Figure 2). In addition to Isochrysis, alkenones are biosynthesized by other haptophyte microalgae, including the widely distributed coccolithophorid Emiliania huxleyi and the closely related species Gephyrocapsa oceanica.18,19 Often these neutral lipids are more abundant than triaclyglycerols, especially in the stationary phase of growth curves.20 Studies by Eltgroth et al. have shown that PULCAs reside in cytoplasmic lipid bodies for energy storage.20 The exact biosynthetic pathways have not been elucidated; however, the proportion of the more unsaturated PULCAs increases with a decreasing water temperature.21,22 These compounds are among the most studied class of lipids in marine organic geochemistry because of their use as proxies for past sea surface temperatures18,23−26 and global atmospheric CO2 concentrations27,28 and, more recently, as indicators for hydrologic,29,30 salinity,31,32 and depositional changes.33 The presence of alkenones in algal-based FAME, their impact on biodiesel fuel

INTRODUCTION On the basis of a resurgence of interest in microalgal-derived biofuels, it is anticipated that many large-scale bioproduction sites will be constructed in the coming decades.1−4 While this field is rapidly changing, the majority of biofuels produced at these facilities will initially be biodiesel, mixtures of fatty acid methyl esters (FAMEs). This substitute for fossil-fuel diesel is produced from reactions between methanol and acylglycerols; the latter are the major components of fats and membrane lipids in algae as well as terrestrial plants. Biodiesel is used to formulate a range of mixtures from B2 (2% biodiesel mixed with 98% fossil-fuel diesel) to B100 (100% biodiesel). Alternatively, “renewable diesel” can be prepared via thermochemical cracking in the presence or absence of a catalyst.5 Inderwildi and King6 stressed the importance of in-depth scientific analysis of biodiesel production, including an advanced chemical prospecting strategy,7,8 to address both economic and ecological requirements. Beyond these aspects, however, potential properties and performance of the fuels based on their composition need to be considered.9 With an interest in identifying ideal species for biodiesel, we investigated the marine prymnesiophyte Isochrysis sp., including strains T-Iso and C-Iso (Figure 1).10,11 We were attracted to Isochrysis sp. because they are farmed commercially for mariculture feedstocks,12−14 and have been cited in reviews on algalbased biofuel.15 Herein, we present a detailed analysis of Isochrysis FAMEs prepared by extraction−transesterification of © 2012 American Chemical Society

Received: January 30, 2012 Revised: March 10, 2012 Published: March 12, 2012 2434

dx.doi.org/10.1021/ef3001828 | Energy Fuels 2012, 26, 2434−2441

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distilled water (10 mL). Once the phases had separated, the bottom layer was drained into a preweighed vial and dried under a stream of N2 and the resulting crude biodiesel was weighed [∼0.9 g, 90% (w/w) of algal oil]. In some cases, the algal oil was spiked with an internal standard, ethyl nonadecanoate, before esterification to evaluate the ability of the method to quantitatively convert an ethyl ester to a methyl ester as well as use the converted methyl ester in the crude product to gauge the recovery of the standard. The reaction was performed without any standards to prepare samples for elemental analysis, 1H nuclear magnetic resonance (NMR) analysis, absorbance spectroscopy, and several types of gas chromatographies. Analysis by One-Dimensional Gas Chromatography with Flame Ionization Detection (GC−FID). FAMEs and alkenones were quantified using a Hewlett-Packard 5890 Series II GC−FID. Compounds were separated on a Supelco SP2380 glass capillary column (60 m length, 0.25 mm inner diameter, and 0.2 μm film thickness) with H2 as the carrier gas. This column was employed specifically for the analysis of FAMEs, in which the elution order differs from typical nonpolar columns (i.e., DB-1). FAMEs were identified with standards purchased from Nu-Chek Prep (Elysian, MN) and Supelco (Bellefonte, PA). Alkenones were identified on the basis of a comparison to the published elution order on gas chromatographic columns, their mass spectra, and mixtures harvested from cultures of Isochrysis sp. In the samples where the algal oil was spiked with ethyl nonadecanoate, methyl nonadecanoate recoveries were >85% and no ethyl nonadecanoate was detected. GC−FID chromatograms of alkenone-spiked B20 mixtures were obtained using a Hewlett-Packard 5890 Series II GC−FID. Samples (1 μL) were injected cool-on-column and separated on a 100% dimethyl polysiloxane capillary column (Restek Rtx-IMS, 30 m length, 0.25 mm inner diameter, and 0.25 μm film thickness) with H2 as the carrier gas at a constant flow of 5 mL/min. The GC oven was programmed from 40 °C (5 min hold) and ramped at 30 °C/min to 120 °C and then at 8 °C/min to 320 °C (5 min hold). Analysis by 1H NMR Spectroscopy. 1H NMR spectra were performed by Chemtos, LLC (Austin, TX). 1H spectra (300 MHz) of Isochrysis algal oil and crude biodiesel were obtained under ambient conditions using CDCl3 as the solvent, which also served as an internal reference (shift value of residual proton at 7.27 ppm). Elemental Analysis. The carbon, hydrogen, and nitrogen (CHN) contents of the dry biomass of Isochrysis sp. before n-hexane extraction, dry biomass after n-hexane extraction, algal oil, and crude biodiesel were determined by Midwest Microlab, LLC (Indianapolis, IN). Absorbance Spectroscopy. The algal oil and crude biodiesel of Isochrysis were green in color, and their pigment content was examined by absorbance spectroscopy. Samples of algal oil and crude biodiesel were dissolved in 100% acetone to a concentration of 100 mg/L. Absorbance between 300 and 800 nm was measured using a ThermoElectron Evolution 300 dual-beam spectrophotometer. Pigment was quantified at 667 nm using specific extinction coefficients for pheophytin.34 Cloud Point (CP) Experiments. To examine the extent of the impact of alkenones on biodiesel cold-flow properties, we prepared and measured the CP of a series of alkenone-spiked biodiesel mixtures. We originally tried to dissolve milligram quantities of purified alkenones35 into a soybean B100 mixture, assuming that high-quality B100 would allow us to assign any changes in CP relative to the alkenone content. Unfortunately, the alkenones would not dissolve in the soybean

Figure 1. Isochrysis galbana (Rt Iso) micrographs: (a) pseudo-colored merge of panels c and d, (b) phase contrast image, (c) Nile Red stained image with 46HE filter (excitation, 500/25; emission, 535/30), and (d) chlorophyll autofluoresence through filterset 50 (excitation, BP640/30; emission, BP690/50). All images were acquired with a Zeiss Plan-Neofluar 40×/0.75 Ph2 objective lens and Zeiss Axiocam MRm monochrome camera.

properties, and the potential for alkenones as a sustainable carbon feedstock have not previously been described.

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EXPERIMENTAL SECTION

Microalgae and Sample Preparation. The marine microalgae Isochrysis sp. “T-iso” used in the present study was obtained from Reed Mariculture (strain CCMP1324) (Santa Cruz, CA). The algae were grown in greenhouse ponds under natural sunlight in a modified F/2 media. Average water temperatures were 18−20 °C. Approximately 2 kg of wet biomass was poured into large crystallizing dishes and freeze-dried. These efforts led to ∼290 g of dry Isochrysis sp. biomass, which was a greenish, dark-brown waxy amorphous solid. Extraction and Quantification of Lipids. The dry Isochrysis sp. biomass obtained above was extracted in 50−150 g batches with n-hexane in a large Soxhlet extraction apparatus. The Soxhlet was allowed to cycle for 48 h (approximately 60 cycles) until the color of the solvent was a faint yellow. Hexane was removed with a rotary evaporator, and the remaining material was transferred to a preweighed vial with dichloromethane and evaporated to dryness with a gentle stream of N2. The weight of the hexane-extractable material (typically ∼10 g from a 50 g dry biomass extraction event) was recorded and will be referred to as “algal oil” following the widely cited protocol of Johnson and Wen.17 This protocol was chosen to allow for a comparison of our results to those of others from different algal species. Acid-Based Esterification of Algal Oil and Production of “Crude Biodiesel”. Following the method of Johnson and Wen,17 a mixture of methanol (3.4 mL), concentrated sulfuric acid (0.6 mL), and chloroform (4.0 mL) was added to 1 g of the algal oil in a 40 mL glass vial containing a small magnetic stir bar. The mixture was then heated to 90 °C while stirring for 40 min. After cooling, the reaction mixture was transferred to a separatory funnel and washed with

Figure 2. Two common alkenone structures produced by Isochyrsis sp., exemplifying a very long carbon chain and the trans double bonds, although studies have found methyl and ethyl alkenones with 35−41 carbons with 2−4 double bonds. The nomenclature for alkenones is similar to that of FAMEs, number of carbons:number of double bonds. 2435


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B100 even with gentle heating. Hoping that fossil-fuel diesel would act as a solvent for alkenones, mixtures of B20 spiked with alkenones were prepared. This approach was successful until efforts to dissolve 4.5 mg of alkenones into 1 mL of B20 failed. CP Analysis. The CP of the fossil-fuel diesel purchased at a local filling station, soybean-derived B100, and the alkenone-spiked B20 blends were analyzed with American Society for Testing and Materials (ASTM) method D2500-09, Standard Test Method for Cloud Point of Petroleum Products. These analyses were performed by Ana Laboratories, Inc. (Newark, NJ).

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RESULTS Extraction of Isochrysis sp. The carbon, hydrogen, and nitrogen contents of the dry Isochrysis sp. biomass were 47.7, 6.95, and 6.26%, respectively (Table 1) (the ash content was Table 1. Isochrysis Dry Biomass Content sample freeze-dried Isochrysis sp. biomass dry biomass after n-hexane extraction algal oil of Isochrysis sp. crude biodiesel of algal oil

carbon (%)

hydrogen (%)

nitrogen (%)

ash (%)

47.7

6.95

6.26

14.9

39.6

6.19

8.41

12.1

74.3 78.2

10.4 11.2

0.82 1.13

Figure 3. Ultraviolet (UV) absorbance spectra of algal oil and crude biodiesel in acetone compared to pheophorbide a. The maxima in the Soret band (410 nm) and Qy band (665 nm) are characteristic of pheophorbide a and pheophytin a, both degradation products of chlorophyll a. The shoulder between 450 and 500 nm in the algal oil spectrum suggests that it may also contain carotenoids. The spectra were scaled to have the same arbitrary Soret band absorbance maximum and offset on the y axis.

14.9%). After extracting with n-hexane,36 the carbon, hydrogen, and nitrogen contents of the remaining biomass were 39.6, 6.19, and 8.41%, respectively. Concentration of the extract by removal of n-hexane in vacuo gave a glossy, dark-green/near-black “grease-like” material that could be handled as a liquid and poured from flask to flask when heated to 40 °C. Following the nomenclature of Johnson and Wen,17 this algal oil was 19.1% of the dry biomass (Reed Mariculture states that the lipid content of their T-iso product is 17%). The carbon, hydrogen, and nitrogen contents of the algal oil were 74.3, 10.4, and 0.82%, respectively. The 1H NMR spectrum of the algal oil was typical of lipid-rich material.37 The absorbance spectrum of the algal oil showed distinct maxima at 410, 505, 534, 609, and 665 nm, which are diagnostic for the chlorophyll degradation products pheophorbide and pheophytin (Figure 3). A red shoulder on the 410 nm was also present, indicating the presence of additional carotenoid pigments. Acid-Catalyzed Esterification of the Algal Oil To Produce a Crude Biodiesel. The acid-catalyzed esterification of the algal oil produced a product that was a dark-green solid at room temperature. Approximately 90% of the algal oil was converted into the crude FAME, in the same range as that obtained by Johnson and Wen,17 as well as several more recent studies.38 The carbon, hydrogen, and nitrogen contents of the crude FAME were 78.2, 11.2, and 1.13%, respectively. 1H NMR analysis showed a strong singlet peak at 3.7 ppm, characteristic for methyl ester protons of the FAMEs created during the esterification of the algal oil.37 The absorbance spectrum was indistinguishable from those of pure pheophorbide and pheophytine (Figure 3). Analysis of Isochrysis Crude Methyl Esters. The lipid profile of the Isochrysis sp. algal oil esterification product indicated that the dominant fatty acid was 18:4n3, with a concentration of 104 mg/g of crude FAME (Table 2). The fatty acid 22:6n-3, docosahexaenoic acid (DHA), was present with a concentration of 74.6 mg/g of crude FAME. Other individual FAMEs are listed in Table 2. The values that we observed were consistent with other studies (Table 3).39,40 Concentrations of

Table 2. Composition of Isochrysis sp. Crude FAME FAMEs C14:0 C16:0 C16:1n-7 C18:0 C18:1n-9 C18:2n-6 C18:3n-6 C18:3n-3 C18:4n-3 C18:5 C20:5n-3 C22:6n-3 total FAMEs total alkenones total FAMEs + alkenones

mg/g of crude FAMEa

mg/g of Isochrysis sp. algal oilb

mg/g of dry weight of Isochrysis sp.

85.0 63.4 35.6 NDc 60.0 26.1 4.75 34.2 115 26 9.7 74.2 533 141 675

75.9 56.7 31.8 ND 53.6 23.3 4.24 30.5 102 23 8.7 12.7 423 126 549

15.9 11.9 6.7 ND 11.3 4.9 0.9 6.4 21.5 4.8 1.8 13.9 76.0 24.2 100

a

Crude FAME is based on the operationally defined method of Johnson and Wen.17 bAlgal oil is the hexane extract of the Isochrysis sp. biomass. cND = not detected.

FAMEs were also calculated on algal oil and dry mass bases. A GC−FID chromatogram for the crude FAME is shown in Figure 4. In addition to FAMEs, the GC−FID chromatogram of the esterification product showed peaks corresponding to polyunsaturated long-chain alkenones (alkenones are also present at 2436


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Table 3. Comparison of the FAMEs (Percentage of Total FAMEs) in the Present Crude FAME Produced by Isochrysis sp. Relative to Two Other Isochrysis sp.37,38 FAMEs

Isochrysis sp. CCMP 1324a

Isochrysis sp. CCMP 1324b

Isochrysis sp. CCMP 1324c

C14:0 C16:0 C16:1n-7 C18:0 C18:1n-9 C18:2n-6 C18:3n-6 C18:3n-3 C18:4n-3 C20:5n-3 C22:6n-3

15.9 11.9 6.7 ND 11.3 4.9 0.9 6.4 21.5 1.8 13.9

10.2 17.6 3.9 ND 32.0 4.1 ND 6.4 15.1 ND 10.9

11.0−22.7 10.2−21.9 ND ND 8.5−29.5 2.0−2.6 ND 2.2−4.5 7.0−19.0 ND 5.3−14.4

Figure 5. CP temperature (°C) versus total alkenone content in B20 mixtures prepared from soybean-derived B100 and fossil-fuel diesel. The B20 mixtures were prepared by dissolving various amounts of alkenones into the fossil-fuel diesel and then adding the soybean B100. Error bars are smaller than symbols.

a

Crude FAME in this study. bData from Liu and Lin.39 cRange produced over four growth phases by Lin et al.40

sample (inset of Figure 6d, 2.25 ppt, w/v), which had a CP of 20 °C.

similar concentrations in the algal oil, indicating that they exist in a free state and are released simply by solvent extraction). This is perhaps not surprising because it is well-known that Isochrysis sp. and some other prymnesiophyte taxa produce these PULCAs,10,21,22 methyl or ethyl ketones with 35−41 carbons, although 37 and 38 carbons are generally the most dominant.41,42 Our Isochrysis sp. crude FAME had an alkenone concentration of 141 mg/g of crude FAME, consisting mainly of the 37:2 and 37:3 alkenones. The total alkenone and FAMEs were 67.5% of the crude product. This value is very similar to that reported by Johnson and Wen17 for the crude FAME yield from Schizochytrium limacinum using either extraction− transesterification (66.4%) or direct transesterification techniques (63.5%, with CHCl3 as the solvent). No free sterols or alcohols were detected, and the nitrogen content in the crude biodiesel likely points to chlorophyll-based pigments. CP Analysis. The CP of the fossil-fuel diesel and soybean B100 used to prepare the B20 mixtures were −9.4 and 2.2 °C, respectively. The B20 mixture had a CP of −6.1 °C, which increased markedly with the alkenone content (Figure 5). In particular for B20 with 0.75−1.13 ppt (w/v) alkenones, the CP temperature jumped from −5 to 2.2 °C, respectively. This is remarkable considering that the GC−FID alkenone signal is barely perceptible even in our highest alkenone-spiked B20

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DISCUSSION To be commercialized, biodiesel must meet a variety of specifications contained in biodiesel standards, such as ASTM D6751 and EN 14214 in Europe. The CP, defined as the temperature at which the first solids become visible when cooling a diesel fuel, is specified as to be reported by ASTM D6751 because this property can be allowed to vary depending upon the time of year and geographic location.43 Similarly, the coldfilter plugging point (CFPP) in EN 14214 is limited by these parameters. Ideally, these values should be low such that the diesel fuel can be used in colder climates. CP criteria are not strictly set by ASTM but rather “reported”. In the case of CP and the related CFPP, ASTM D975 provides guidance for operation temperatures based on 10th percentile minimum temperatures.44 Melting points of fatty esters are a useful parameter to assess the suitability of individual components regarding the lowtemperature properties of biodiesel.45 In general, melting points increase with increasing carbon length and decrease with increasing unsaturation (assuming cis-alkene geometry). For this reason, certain classes of compounds, such as those with trans double bonds, are undesirable in biodiesel.46 Others include

Figure 4. GC−FID chromatogram of the crude FAME produced from Isochrysis sp. biomass. The respective number of carbons for FAMEs and alkenones are labeled along the x axis. 2437


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Figure 6. Chromatograms of B20 solutions (80% fossil-fuel diesel and 20% B100 from soybean oil) prepared for CP analysis: (a) B20, (b) B20 with alkenones (0.11, w/v), (c) B20 with alkenones (0.75, w/v), and (d) B20 with alkenones (2.25, w/v). Except for the FAME (C16 and C18) content indicated in the chromatogram, compounds in the first 20 min are fossil-fuel diesel constituents. C14 FAME coelutes with fossil-fuel peaks and is annotated.

cis-monounsaturated esters longer than C18 (melting points between approximately −8 and 9 °C) and saturated esters longer than C10 (methyl undecanoate, with a melting point = −12 °C). An analysis of the FAME profile of our Isochrysis methyl esters indicates that methyl myristate (C14:0) and methyl palmitate (C16:0) account for a significant proportion of the FAMEs (17.1 and 12.8%, respectively; see Table 3). We speculated, however, that the obvious cold-flow problems with Isochrysis crude FAME (i.e., solid at room temperature) could

be equally explained by their high alkenone content [21% (w/w) of algal oil; see Table 2]. For example, the CP of palm oil methyl esters with >40% methyl palmitate is around 16 °C;47 therefore, the obviously even higher CP of Isochrysis crude FAME must be explained by the presence of other components. Unlike unsaturated FAMEs that have methylene-interrupted cis (Z) double bonds, alkenones have trans (E) double bonds separated by five and occasionally three methylene groups.40,41,48−50 Their long chain length and trans double bond geometry results 2438


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As shown in Figure 4, these compounds may elute beyond the maximum retention time and temperature gradient of a standard GC−FID analysis for biodiesel and biodiesel blends. While Hu et al. summarized the presence of many very longchain fatty acids (>C20) of several algae (including haptophyta Emiliania huxleyi and I. galbana) and cyanobacteria,3 PULCAs were not considered by this group. Synthesis of PULCAs has been documented to be restricted taxonomically within the haptophyte order Isochrysidales.21,55 Consequently, these algae might be avoided for biodiesel unless downstream refining, such as crystallization fractionation, is performed.56,57 However, the otherwise positive attributes of Isochrysis sp. noted earlier may be more ideal for generating other biofuels, such as methane from catalytic gasification of biomass, which would be less dependent upon the lipid composition but more focused on lipid production of algae.58,59 Alkenones themselves arguably represent a promising renewable carbon resource. Their structures are particularly well-suited for catalytic cracking, a process that historically has been used in the petroleum industry for the conversion of less desirable heavy distillates to lower molecular-weight commodity chemicals and fuels60,61 and has more recently appeared in biofuel technologies.62 Specifically, alkenones are non-aromatic with long olefinic carbon chains, have favorable carbon/ hydrogen ratios, and have few heteroatoms.63−65 There is no prior work on the large-scale production of alkenones from Isochrysis sp. From our results, however, we can calculate a total alkenone content of dry biomass to be approximately 5% (24.2% of the 19.1% total lipid content; see Table 2). A review of algae for biodiesel listed 175 tons of biomass dry weight hectare−1 year−1 for I. galbana (outdoor pond).54 This would correspond to nearly 9 tons of total alkenones hectare−1 year−1, a not insubstantial amount of untapped renewable carbon from an attractive bioresource. The sum of FAMEs and alkenones accounted for approximately 53 and 14%, respectively, of the mass of the crude biodiesel. Pigment analysis suggests that pheophorbide and chlorophyll degradation products account for an additional 5% of the mass, leaving a substantial fraction of the mass unaccounted. Johnson and Wen similarly reported that FAMEs accounted for 66% of the mass of crude biodiesel from S. limacinum;17 this organism is not thought to produce alkenones. A mass balance calculation indicates that the remaining 28% of the mass of the crude biodiesel that is not FAMEs, alkenones, or pheophorbides is composed of material that is 76% carbon and 2% nitrogen. Thus, this unaccounted material is substantially poorer in nitrogen than bulk crude biodiesel or algal oil (Table 1), ruling out substantial contributions from classes of potentially hydrolysis-resistant molecules, such as sphingolipids, porphyrins, or lipopeptides. Our investigations continue, aimed at characterizing this remaining material and evaluating its potential as a carbon-rich industrial feedstock.

in relatively high melting points (pure C36:2 alkenone has a melting point of 58 °C).51 Imahara and co-workers demonstrated that even minor amounts of higher melting components can have a dramatic impact on biodiesel coldflow properties.52 Specifically, the addition of small amounts of methyl palmitate (C16:0) or methyl stearate (C18:0) to representative biodiesel mixtures greatly increased CP values. Importantly, irrespective of the biodiesel composition, mixtures with fixed C16 or C18 saturated ester content had nearly identical CPs. Our alkenone-spiked biodiesel mixtures provide further evidence that CP is largely determined by the abundance of components with higher melting temperatures. Indeed, our casual laboratory observations illustrate the relative insolubility and highly nonpolar nature of alkenones. For instance, while pure C36:2 alkenone could be dissolved in dichloromethane and benzene, it was only partially soluble in ethereal solvents and completely insoluble in more polar solvents, such as acetone and methanol. The solubility observations themselves show that alkenones likely cause cold-flow problems. Some other specifications may not be significantly affected by alkenone contamination. For example, kinematic viscosity in most cases is directly proportional to the content of the individual components, so that minor amounts of alkenones likely would not cause this specification to be exceeded. The cetane number may also be acceptable because of the long hydrocarbon chain in the alkenones offsetting the cetane-lowering effect of the double bonds and the observation, similar to kinematic viscosity, that the cetane number is generally proportional to the content of the individual components. However, cold flow is likely not the only technical problem that FAME from Isochrysis sp. would face. The significant amounts of polyunsaturated fatty acids (PUFAs), especially 18:3n-3 and 22:6n-3, would likely cause poor oxidative stability. The oxidative stability specifications per the so-called Rancimat test in the biodiesel standards ASTM D6751 and EN 14214 are 3 and 6 h, respectively. However, even monounsaturated methyl oleate does not meet this specification in the neat form (2.79 h), much less the polyunsaturated methyl linoleate (0.94 h) and methyl linolenate (0 h).45 The PUFA with four or more double bonds in Isochrysis sp. would therefore exhibit similar poor oxidative stability, affecting the oxidative stability of the FAME mixture. It may also be noted that the European biodiesel standard EN 14214 does not permit more than 1% of PUFA with greater than three double bonds. Our absorbance spectroscopy data indicate that pheophorbide and related chlorophyll degradation products account for approximately 5% (w/w) of the crude FAME. Pigments of this type have also been shown to adversely affect biodiesel oxidative stability.53 Otherwise, Isochrysis sp. is already grown commercially for mariculture; therefore, many of the hurdles associated with large-scale biodiesel production have been overcome (we also analyzed one commercial mariculture product (T-Isochrysis Algae Paste, 10; www.brineshrimpdirect.com) and found that it had a similar distribution of FAMEs and alkenones). Moreover, in one study of 55 microalgal species for biodiesel production, Isochrysis galbana proved to be average in both biomass yield and lipid productivity.54 The average lipid content in 15 nutrientreplete and nitrogen-deficient cultures was 25 and 29% dry weight, respectively. Therefore, Isochrysis sp. is attractive, despite alkenone contamination and a problematic fatty acid profile. However, even minor feedstock components containing alkenones (levels above 1 ppt; Figure 4) can affect fuel quality.

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CONCLUSION In summary, marine algae have promise for the production of biodiesel. Isochrysis sp. is attractive under the aspects of favorable growth properties and history of commercial mariculture. In addition to FAMEs, this species of microalgae produces a significant amount of long-chain alkenones; these had not previously been described in connection with algalbased biofuels. Alkenones represent a significant proportion of the Isochrysis total lipid extract (TLE), are unaffected by 2439


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(14) Enright, C. T.; Newkirk, G. F.; Craigie, J. S.; Castell, J. D. J. Exp. Mar. Biol. Ecol. 1986, 96, 1−13. (15) For recent reviews that include Isochrysis, see: (a) Mata, T. M.; Martins, A. A.; Caetano, N. S. Renewable Sustainable Energy Rev. 2010, 14, 217−232. (b) Huerlimann, R.; de Nys, R.; Heimann, K. Biotechnol. Bioeng. 2010, 107, 245−257. (c) Singh, J.; Gu, S. Renewable Sustainable Energy Rev. 2010, 14, 2596−2610. (16) Nagle, N.; Lernke, P. App. Biochem. Biotechnol. 1990, 24, 355−361. (17) Johnson, M. B.; Wen, Z. Energy Fuels 2009, 23, 5179−5183. (18) Volkman, J. K.; Eglinton, G.; Corner, E. D. S. Phytochemistry 1980, 19, 2619−2622. (19) Marlowe, I. T.; Brassell, S. C.; Eglinton, G.; Green, J. C. Org. Geochem. 1984, 6, 135−141. (20) Eltgroth, M. L.; Watwood, R. L.; Wolfe, G. V. J. Phycol. 2005, 41, 1000−1009. (21) Volkman, J. K.; Barrerr, S. M.; Blackburn, S. I.; Sikes, E. L. Geochim. Cosmochim. Acta 1995, 59, 513−520. (22) Conte, M. H.; Thompson, A.; Lesley, D.; Harris, R. P. Geochim. Cosmochim. Acta 1998, 62, 51−68. (23) Brassell, S. C.; Eglinton, G.; Marlowe, I. T.; Pflaumann, U.; Sarnthein, M. Nature 1986, 320, 129−133. (24) Prahl, F. G.; Wakeham, S. G. Nature 1987, 330, 367−369. (25) Eglinton, G.; Bradshaw, S. A.; Rosell, A.; Sarnthein, M.; Pflaumann, U.; Tiedemann, R. Nature 1992, 356, 423−426. (26) Müller, P. J.; Kirst, G.; Ruhland, G.; von Storch, I.; Rosell-Melé, A. Geochim. Cosmochim. Acta 1998, 62, 1757−1772. (27) Jasper, J. P.; Hayes, J. M. Nature 1990, 347, 462−464. (28) Jasper, J. P.; Hayes, J. M.; Mix, A. C.; Prahl, F. G. Paleoceanography 1994, 9, 781−798. (29) Pahnke, K.; Sachs, J. P.; Keigwin, L. D.; Timmerman, A.; Xie, S.-P. Paleoceanography 2007, 22, PA4214. (30) Van der Meer, M. T. J.; Bass, M.; Rijpstra, I. C.; Marino, G.; Rohling, E. J.; Sinninghe, D. J. S.; Schouten, S. Earth Planet. Sci. Lett. 2007, 262, 594−600. (31) Schouten, S.; Ossebaar, J.; Schreiber, K.; Kienhuis, M. V. M; Langer, G.; Benthien, A.; Bijma, J. Biogeosciences 2006, 3, 113−119. (32) Sachs, D.; Sachs, J. P. Geochim. Cosmochim. Acta 2008, 72, 793−806. (33) Englebrecht, A. C.; Sachs, J. P. Geochim. Cosmochim. Acta 2005, 69, 4253−4265. (34) Lorenzen, C. J.; Jeffrey., S. W. UNESCO Tech. Pap. Mar. Sci. 1980, 35, 20. (35) Alkenones were purified according to: Ohkouchi, N.; Xu, L.; Reddy, C. M.; Montluçon, D.; Eglinton, T. I. Radiocarbon 2005, 47, 401−412. (36) Extraction was also performed using 1:2 (v/v) CH2Cl2/MeOH as described in: Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911−917. For a comparison of the FAME/alkenone profile following transesterification of this material, see the Supporting Information. (37) See the Supporting Information. (38) For select examples, see: (a) D’Oca, M. G. M.; Viêgas, C. V.; Lemões, J. S.; Miyasaki, E. K.; Morόn-Villarreyes, J. A.; Primel, E. G.; Abreu, P. C. Biomass Bioenergy 2011, 35, 1533−1538. (b) Cheng, C.-H.; Du, T.-B.; Pi, H.-C.; Jang, S.-M.; Lin, Y.-H.; Lee, H.-T. Bioresour. Technol. 2011, 102, 10151−10153. (39) Liu, C. P.; Lin, L. P. Bot. Bull. Acad. Sin. 2001, 42, 207−214. (40) Lin, Y. H.; Chang, F. L.; Tsao, C. Y.; Leu, J. Y. Biochem. Eng. J. 2007, 37, 166−176. (41) Rontani, J.-F.; Marchand, D.; Volkman, J. K. Org. Geochem. 2001, 32, 1329−1341. (42) Rontani, J.-F.; Prahl, F. G.; Volkman, J. K. J. Phycol. 2006, 42, 800−813. (43) American Society for Testing and Materials (ASTM). ASTM Standard D6751, Standard Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels; ASTM International: West Conshohocken, PA, 2003; DOI: 10.1520/D6751-11B, www.astm.org. (44) American Society for Testing and Materials (ASTM). ASTM Standard D975, Standard Specification for Diesel Fuel Oils; ASTM

transesterification, remain a component of the resulting biodiesel, and detrimentally affect biodiesel fuel quality. Efforts are ongoing to better understand the parameters that effect alkenone production in Isochrysis and content in biodiesel mixtures, along with investigations into their conversion to high-value commodity chemicals and fuels.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of Isochrysis algal oil and crude FAME and the FAME/alkenone profile of crude FAME produced using the Bligh and Dyer36 extraction method. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: (360) 650-6283. Fax: (360) 650-2826. E-mail: oneil@ chem.wwu.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the following for their generous support of this research: Western Washington University, M. J. Murdock Charitable Trust and Research Corporation Departmental Development Grant, the Hollister Fund and Lowell Foundation at WHOI, the Croucher Foundation and Lucy B. Lemann Research Award at MBL, Rodney Berens, the Charles D. Hollister Endowed Fund for Innovative Research, the Hollis and Ermine Lovell Charitable Foundation, Pancha and Carl Peterson, Elizabeth and James Pickman, the Kathleen and Peter Naktenis Foundation, Frank Mittricker, and Massachusetts Clean Energy Center. We thank Gary Wikfors from the NOAA/NMFS Northeast Fisheries Center in Milford, CT, who provided us with the starter algal cultures. We thank Dr. John Volkman for his advice and comments during previous drafts of this manuscript.

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REFERENCES

(1) Chisti, Y. Biotechnol. Adv. 2007, 25, 294−306. (2) Cockerill, S.; Martin, C. Biotechnol. Biofuels 2008, 1, 1−6. (3) Hu, Q.; Sommerfeld, M.; Jarvis, E.; Ghirardi, M.; Posewitz, M.; Seibert, M.; Darzins, A. Plant J. 2008, 54, 621−639. (4) Huntley, M.; Redalje, D. Mitigation Adapt. Strategies Global Change 2007, 12, 573−608. (5) Knothe, G. Prog. Energy Combust. Sci. 2010, 36, 364−373. (6) Inderwildi, O. R.; King, D. A. Energy Environ. Sci. 2009, 2, 343−346. (7) Eisner, T. Issues Environ. Sci. Technol. 1990, 6, 31−34. (8) Eisner, T. Chemical prospecting: A proposal for action. In Ecology, Economics, Ethics: The Broken Circle; Bormann, F. H., Kellert, S. R., Eds.; Yale University Press: New Haven, CT, 1991; pp 196−202. (9) Knothe, G. Green Chem. 2011, 13, 3048−3065. (10) Patterson, G. W.; Tsitsa-Tsardis, E.; Wikfors, G. H.; Gladu, P. K.; Chitwood, D. J.; Harrison, D. Phytochemistry 1994, 35, 1233− 1236. (11) Wikfors, G. H.; Patterson, G. W. Aquaculture 1994, 123, 127−135. (12) Lavens, P.; Sorgeloos, P. Manual on the Production and Use of Live Food for Aquaculture; Food and Agriculture Organization of the United Nations: Rome, Italy, 1996; Fisheries Technical Paper 361. (13) Albentosa, M.; Pérez-Camacho, A.; Labarta, U.; Fernández-Reiriz, M. J. Aquaculture 1996, 148, 11−23. 2440


Energy & Fuels

Article

International: West Conshohocken, PA, 2003; DOI: 10.1520/D097511, www.astm.org. (45) The Biodiesel Handbook, 2nd ed.; Knothe, G., Krahl, J., Gerpen, J. V., Eds.; American Oil Chemists’ Society (AOCS) Publishing: Urbana, IL, 2010. (46) Knothe, G. Energy Fuels 2008, 22, 1358−1364. (47) Benjumea, P.; Agudelo, J.; Agudelo, A. Fuel 2008, 87, 2069− 2075. (48) Marlowe, I. T.; Green, J. C.; Neal, A. C.; Brassell, S. C.; Eglinton, G.; Course, P. A. Eur. J. Phycol. 1984, 19, 203−216. (49) Rechka, J. A.; Maxwell, J. R. Tetrahedron Lett. 1988, 29, 2599− 2600. (50) Rechka, J. A.; Maxwell, J. R. Org. Geochem. 1988, 13, 727−734. (51) O’Neil, G. W.; Moser, D. J.; Volz, E. O. Tetrahedron Lett. 2009, 50, 7355−7357. (52) Imahara, H.; Minami, E.; Saka, S. Fuel 2006, 85, 1666−1670. (53) Issariyakul, T.; Dalai, A. K. Energy Fuels 2010, 24, 4652−4658. (54) Griffiths, M. J.; Harrison, S. T. L. J. App. Phycol. 2009, 21, 493−507. (55) Conte, M. H.; Volkman, J. K.; Eglinton, G. Lipid biomarkers of the Haptophyta. In The Haptophyte Algae; Green, J. C., Leadbetter, B. S. C., Eds.; Clarendon Press: Oxford, U.K., 1994; Vol. 51, pp 351−377. (56) Lee, I.; Johnson, L. A.; Hammond, E. G. J. Am. Oil Chem. Soc. 1996, 73, 631−636. (57) Kerschbaum, S.; Rinke, G.; Schubert, K. Fuel 2008, 87, 2590− 2597. (58) Haiduc, A. G.; Brandenberger, M.; Suquet, S.; Vogel, F.; Bernier-Latmani, R.; Ludwig, C. J. App. Phycol. 2009, 21, 529−541. (59) Stucki, S.; Vogel, F.; Ludwig, C.; Haiduc, A. G.; Brandenberger, M. Energy Environ. Sci. 2009, 2, 535−541. (60) DeCroocq, D. Catalytic Cracking of Heavy Petroleum Fractions; Editions Technip: Paris, France, 1984. (61) Peters, A. W.; Flank, W. H.; Davis, B. H. The history of petroleum cracking in the 20th century. In Innovations in Industrial and Engineering Chemistry; Flank, W. H., Abraham, M. A., Matthews, M. A., Eds.; American Chemical Society (ACS): Washington, D.C., 2008; pp 103−187. (62) For a review, see: Taufiqurrahmi, N.; Bhatia, S. Energy Environ. Sci. 2011, 4, 1087−1112. (63) Lerner, B. A.; Himpsl, F. L. Oil Gas J. 1997, 95, 61. (64) Bollas, G. M.; Vasalos, I. A. Ind. Eng. Chem. Res. 2004, 43, 3270−3281. (65) Shi, T. P.; Xu, Z. M.; Cheng, M.; Hu, Y. X.; Wang, R. A. Energy Fuels 1999, 13, 871−876.

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