This work was supported by the National Science Foundation (CHE-1151492), the Northwest Advanced Renewables Alliance (fellowship to J. Wilson-Peltier), and through a private donation from friends of WHOI. We thank Kevin R. Steidley and Kim Ascherl (USDA / ARS / NCAUR) for excellent technical assistance.

1. Microalgae and Biomass Preparation
Note: The marine microalgae Isochrysis sp. &#34;T-iso&#34; used in the present study can be purchased (see List of Materials). Multi-kilogram quantities of Isochrysis can be purchased as a frozen wet paste (Iso-paste) containing approximately 80% water and 20% biomass, and is dark green/near black in color with a pungent odor smelling of the sea. Isochrysis can also be purchased as a dry (95% dry) yellow-brown powder (Iso-powder) with a similar odor.
<ol>
	<li>In order to dry the Isochrysis paste, open a 1 kg package by cutting a 1 - 2 inch hole in the corner of the plastic packaging using scissors.</li>
	<li>Squeeze approximately 300 g of this Isochrysis paste through the hole into a 150 mm x 75 mm crystallizing dish to create a thin layer (~20 mm).</li>
	<li>Leave the paste to air-dry at room temperature until it becomes dry and flaky (typically, 48 - 96 hr). 
	Note: Actual drying times may vary and are dependent on temperature. However, no difference was noticed in the yields or product quality with even longer drying times (up to two weeks). The drying process may also be made more uniform and/or accelerated by placing the crystallizing dish on a warm plate (30 - 40 &#176;C).</li>
	<li>Scrape the dry biomass from the crystallizing dish using a spatula and collect into a cellulose extraction thimble (Length: 123 mm, 43 mm ID). Record the weight of the dry Isochrysis biomass.</li>
</ol>
2. Soxhlet Extraction of Dry Isochrysis Biomass
<ol>
	<li>Load an Isochrysis-containing cellulose extraction thimble (typically 50 - 60 g dry biomass) into a Soxhlet extraction apparatus.</li>
	<li>Fill the Soxhlet flask with hexanes (400 ml), turn on the condenser water and heat source, and allow the Soxhlet to cycle for 24 - 48 hr (until the color of the solvent has gone from dark green to a faint yellow).</li>
	<li>Turn off the heat and allow the apparatus to cool to room temperature, then disconnect the flask from the Soxhlet extractor.</li>
	<li>Remove the hexanes using a rotary evaporator and record the weight of the hexanes-extractable material (&#34;hexane algal oil&#34; (h-AO)).</li>
</ol>
3. Saponification of the Algal Oil and Separation of Fatty Acids and Neutral Lipids
<ol>
	<li>Redissolve the h-AO in the same round bottom flask from Step 2.4 above with methanol:dichloromethane (2:1, volume = 10 x mass of algal oil).</li>
	<li>Add a stir bar and attach a reflux condenser (Coil: 500 mm length).</li>
	<li>Add H2O (volume = 2.67 x mass of algal oil) and KOH (50% w/w algal oil) and heat the contents with stirring to 60 &#176;C for 3 hr.</li>
	<li>After cooling to room temperature, remove the organic solvents (methanol and dichloromethane) on a rotary evaporator.</li>
	<li>Transfer the remaining aqueous mixture by pouring into a 1-L separatory funnel. Add hexanes (equivalent in volume to the aqueous solution), shake the separatory funnel, and allow the layers to separate.</li>
	<li>Drain the lower aqueous layer into an Erlenmeyer flask, and pour off the top organic phase into a separate Erlenmeyer.</li>
	<li>Repeat steps 3.5 and 3.6 until the organic layer is colorless (typically 1-2 more times).</li>
	<li>Concentrate the combined organic extracts on a rotary evaporator to isolate the neutral lipids as a greenish solid (mp. &#8776; 60 - 70 &#176;C).</li>
	<li>Acidify the aqueous phase with HCl (6 M, until pH ~ 2 as indicated by pH paper).</li>
	<li>Extract the free fatty acids (FFAs) from the acidified aqueous phase with hexanes (equivolume to the aqueous phase) using a 1 L separatory funnel as described in steps 3.5 and 3.6.</li>
	<li>Remove the hexanes on a rotary evaporator to obtain the FFAs as a dark green near-black oily residue (liquid at temperatures &#62;30 &#176;C).</li>
</ol>
4. Acid-catalyzed Esterification of Free Fatty Acids and Production of a Green Biodiesel
<ol>
	<li>Transfer the FFAs using methanol:chloroform (1:1, 6 x volume of algal oil) to first dissolve the FFAs and then by pouring into a thick-walled high pressure reaction flask equipped with a stir bar.</li>
	<li>Add concentrated H2SO4 (20% w/w algal oil), seal the flask, and heat the mixture to 90 &#176;C while stirring for 1 hr.</li>
	<li>After cooling to room temperature, transfer the mixture by pouring into a separatory funnel.</li>
	<li>Add H2O (2 x volume of algal oil), shake the separatory funnel, and allow the phases to separate.</li>
	<li>Drain the bottom layer into a pre-weighed round bottom flask and concentrate on a rotary evaporator. Record the mass of the resulting biodiesel.</li>
	<li>Analyze the fatty acid profile by gas chromatography with flame-ionization detector (GC-FID)28 (gas chromatograph equipped with a DB-88 [(88% cyanopropyl) methylarylpolysiloxane] column (30 m x 0.25 mm ID x 0.20 &#181;m film thickness). 
	Note: Common fatty acid methyl esters are verified by retention time comparison with authentic samples obtained commercially. Additionally, gas chromatography-mass spectrometry (GC-MS; gas chromatograph coupled to a mass selective detector) is performed under identical conditions of temperature program and column to analyze components such as C18:4 for which no authentic standards are available with the results applied to GC quantitation.</li>
</ol>
5. Biodiesel Decolorization
<ol>
	<li>Heat the dark green colored biodiesel to 60 &#176;C in a round bottom flask equipped with a stir bar.</li>
	<li>Add montmorillonite K 10 (MK10) powder (10-20% w/w of the biodiesel) and stir for 1 hr.</li>
	<li>Remove the round bottom from the heat and allow the solution to cool to room temperature.</li>
	<li>Prepare a filtration apparatus consisting of a round bottom flask and filter funnel containing a cellulose filter paper (Ash 0.007%).</li>
	<li>Pour the cooled decolorized biodiesel through the filter funnel using a minimal amount of hexanes to rinse the round bottom flask.</li>
	<li>Disconnect the filter funnel from the round bottom flask (this now contains a hexanes solution of the decolorized biodiesel) and remove the hexanes with a rotary evaporator to afford an orange/red biodiesel.</li>
	<li>Store samples at 4 &#176;C during which time some settling of insoluble material (~10% w/w) will occur.</li>
	<li>Remove the insoluble material by decanting or filtration as described in step 5.4 and 5.5 to produce a clear homogeneous biodiesel for analysis.</li>
</ol>
6. Isolation and Purification of Alkenones from the Neutral Lipids
<ol>
	<li>Dissolve the neutral lipids (from Step 3.8) in a minimal amount of dichloromethane (approximately 50 ml for 10 g neutral lipids) and add the solution with a pipet to the top of a chromatography column (O.D 60 mm, ID 55 mm, length 18&#34;) containing silica gel (230-400 mesh, 100 g).</li>
	<li>Elute the solution through the silica with pressure (~5 psi) using dichloromethane (approximately 150 ml) as solvent and collect the eluent in a 250 ml round bottom flask.</li>
	<li>Remove the dichloromethane with a rotary evaporator to give an orange-colored solid.</li>
	<li>Recrystallize the solid using hexanes by adding approximately 100 ml of boiling hexanes followed by incremental additional amounts of boiling hexanes until the solution is homogeneous (total volume ~ 150 ml). Then, slowly cool the solution to room temperature to promote crystallization.</li>
	<li>Collect the crystallized alkenones using a filtration apparatus as described in step 5.4 using a small amount of cold (0 &#176;C) hexanes to rinse the flask.</li>
</ol>

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</ol>

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Isochrysis is one of only a select number of algal species farmed industrially, harvested as a primary component of shellfish feed, and therefore representative of the scale necessary for biofuel production. The availability of the algae utilized and standard methods employed in this study, make the protocol presented widely accessible to other groups for further investigations. Critical steps include air-drying the algae (as opposed to lyophilizing33), solvent extractions, saponification, and esterification. Through these operations one can examine yields of lipids and other coproducts from the various Isochrysis available30. It is anticipated that these may differ as a result of differing strains and cultivation methods,31 and may also be impacted by the nature of the product and any additional processing (e.g., drying or freezing) utilized by the supplier. As we demonstrate here, the protocol developed can be successfully applied to different types of Isochrysis products, ranging from a wet paste to a dry milled powder. Yields of biodiesel were however lower from the powdered biomass (7% w/w dry biomass vs. 12% w/w from the dried paste), which corresponds with lower amounts of algal oil (h-AO) extracted. This may suggest that an alternative extraction protocol32 other than a Soxhlet apparatus may be better suited for dry powdered Isochrysis products. The Isochrysis powder used in this study is advertised as containing 23-25% lipids, which similar to what we have experimentally obtained from dried Isochrysis paste.33,34,26
Despite the different colors of the starting dry biomass, the Iso-paste-hAO and Iso-powder-hAO were essentially indistinguishable, both dark green/near black solids with melting points of approximately 50 &#176;C. Interestingly, the ratio of FFAs to neutral lipids within the two hexane extracts was different. After saponification and separation of the neutral lipids, we obtained 60% (w/w) FFAs and 40% (w/w) neutral lipids from the Iso-paste-hAO. The Iso-powder-hAO produced on average 46% (w/w) FFAs and 54% (w/w) neutral lipids. The results suggest that either the starting powdered biomass may contain higher amounts of neutral lipids relative to FA derivatives than the Isochrysis paste, or that Soxhlet extraction of powdered Isochrysis is somewhat selective for neutral lipids.
Not only were the yields of products obtained from the two commercial Isochrysis biomasses different, but also the fatty acid profiles of the resulting biodiesel. This is important, as the fuel properties of a biodiesel are directly dependent on the nature and contents of individual FAMEs.35 To be commercialized, all biodiesel must conform to the standards described in the documents ASTM D6751 or EN 14214 in the U.S or Europe respectively. Specifications include ranges for lubricity and kinematic viscosity, and minimum values for cetane number and oxidative stability. Other important recommendations are related to cold flow properties in the form of a cloud point (CP) or cold filter plugging point (CFPP). We have previously reported results from the comprehensive fuel testing of biodiesel prepared from Iso-paste.36 Since the FAME profile of biodiesel produced from the Iso-powder in this study is similar to those previously tested, we can predict certain fuel properties to be similar for both biodiesel fuels. For instance, polyunsaturated fatty acids (PuFAMEs, more than two double bonds) account for approximately 40% of both FAME mixtures (35.2% and 39.9%, Table 2). This will result in poor oxidative stability and favorable cold-flow.35 There are, however, slight differences in the FAME profiles of the two biodiesel fuels. Biodiesel produced from the powdered Isochrysis contained higher amounts of 14:0 (19.4 mg/g vs. 16.4 mg/g), 18:3 (13.5 mg/g vs. 8.5 mg/g), and 22:6 (11.0 mg/g vs. 6.9 mg/g) FAMEs, yet lower amounts of 18:4 (10.4 mg/g vs. 19.8 mg/g). The extent of the impact of these differences on the various fuel properties contained in the ASTM standards remains to be investigated.
The initial biodiesel obtained from both commercial Isochrysis algae were similarly dark green in color which can be explained by the presence of chlorophylls.36 Chlorophyll and its derivatives have been reported to have a negative effect on the stability of vegetable oils and their corresponding biodiesel fuels.36,29 Based on the method of Issariyakul and Dalai for decolorizing greenseed canola oil in connection with biodiesel production,29 stirring our green biodiesel over 10% (w/w) MK10 at 60 &#176;C for 1 hr resulted in a dramatic reduction in pigment content by visual inspection (ref. Figure 2). Mass recoveries from the decolorization process were on average 90%.
Yields of purified alkenones from Iso-paste-hAO and Iso-powder-hAO neutral lipids were comparable at 40% and 46% w/w respectively (Table 1). Since neutral lipids represent a higher proportion of material contained in the Iso-powder-hAO (54% w/w vs. 40% w/w), alkenone yield from the Iso-powder-hAO exceeds the Iso-paste-hAO alkenone yield by approximately 10% (25% w/w vs. 16% w/w). However, considering that yields of the Iso-powder-hAO itself were lower than Iso-paste-hAO (15% vs. 20% w/w), overall yields of alkenones from both dry Isochrysis biomasses are more similar (0.2 x 0.4 x 0.4 = 3.2% w/w from dried Isochrysis paste and 0.15 x 0.54 x 0.46 = 3.7% from Isochrysis powder).

There has recently been a great resurgence of interest in biofuels from algae, particularly for the production of liquid fuels such as biodiesel1 and other biomass-derived oils.2 Proposed benefits include the avoidance of certain food vs. fuel controversies3 and reportedly higher productivities and CO2 mitigation capabilities than traditional agricultural crops.4 This follows the nearly 20 year United States Department of Energy&#39;s Aquatic Species Program (ASP) started in 1978 for the purpose of investigating transportation fuel from algae. As outlined in Sheehan&#39;s report,5 the program ended in 1996 primarily because projected costs were not competitive with crude petroleum at that time ($18.46 per barrel (159 L)). While the cost of petroleum has increased dramatically since then ($87.39 per barrel in 2014)6, which is connected to the renaissance in algal biofuel research, some have argued that nonetheless algal biofuels will prove too costly.7 As one strategy to offset biofuel production costs, the notion of value-added coproducts has emerged among both critics7,8 and proponents9,10 and features as one of the key reasons for pursuing algal biofuels in the United States Department of Energy (DOE) &#34;National Algal Biofuels Technology Roadmap&#34;.11 
Here we describe a method for the coproduction of two separate fuel streams from commercial Isochrysis microalgae. We have focused on Isochrysis in part because it is already produced industrially, harvested for purposes of mariculture, and also because Isochrysis is one of only a few species of algae that in addition to traditional lipids (i.e., fatty acids) biosynthesize a unique class of compounds known as polyunsaturated long-chain alkenones.12 Alkenone structures are characterized by very long hydrocarbon chains (36-40 carbons), two to four non-methylene interrupted trans-double bonds, and a methyl or ethyl ketone (Figure 1). Alkenone unsaturation is sensitive to the algae growing temperature,13,14 such that the proportion of the diunsaturated C37 methyl alkenone (the so-called &#34;unsaturation index&#34;) can be used as a proxy for past sea surface temperatures.15&#8722;20 Alkenones are thought to reside in cytoplasmic lipid bodies and can be more abundant than triglycerides (TAGs).21,22 Under nitrogen or phosphorous limitation, up to 10&#8722;20% of cell carbon in the stationary phase is accumulated as alkenones.23,24 From an evolutionary standpoint, alkenones may have been favored over TAGs because their trans-double bond geometry provides a more stable form of energy storage.21
<img alt="Figure 1" src="/files/ftp_upload/54189/54189fig1.jpg" /> 
Figure 1. Structures of polyunsaturated long-chain alkenones. Common alkenone methyl 37:3 isolated from Isochrysis exemplifying long hydrocarbon chain lengths (36 - 40 carbons), trans non-methylene interrupted double bonds, and terminating in a methyl or ethyl ketone. Nomenclature is similar to fatty acids where #:# refers to number of carbons:number of double bonds. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54189/54189fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We argue that alkenones represent a promising renewable carbon feedstock from a common algae with a history of industrial cultivation.25 Biodiesel produced directly from the total lipid extract of Isochrysis contains a significant amount (10-15% w/w) of alkenones and contamination by these high-melting compounds results in poor cold flow fuel properties. However, using the saponification/extraction techniques described here, alkenones can be removed and recovered thereby improving the biodiesel quality while producing a secondary product stream. Recently we demonstrated the conversion of alkenones to a liquid fuel by cross-metathesis with 2-butene (butenolysis).26 The butenolysis reaction employs a commercial ruthenium metathesis-initiator, occurs rapidly at low temperature, and cleanly delivers a predictable mixture of jet fuel range hydrocarbons. This reaction is performed in parallel with biodiesel synthesis from fatty acids, representing the first steps toward a &#34;biorefinery&#34; approach27 for commercially viable Isochrysis biofuel production.

<table><tbody><tr><td>&lt;em&gt;Isochrysis&lt;/em&gt;</td><td>Reed Mariculture</td><td>Iso, Raw, Unprocessed, 20%dw</td><td>Live culture paste, 20% w/w biomass</td></tr><tr><td>&lt;em&gt;Isochrysis&lt;/em&gt;</td><td>Necton, S.A</td><td>AADISS004</td><td>PhytoBloom Prof Isochrysis (Freeze-dried)</td></tr><tr><td>Hexanes</td><td>Fisher Chemical</td><td>H291-4</td><td>ACS Certified</td></tr><tr><td>Methanol</td><td>Fisher Chemical</td><td>A452-4</td><td>HPLC Grade</td></tr><tr><td>Dichloromethane</td><td>Fisher Chemical</td><td>D37-4</td><td>Certified/Stabilized</td></tr><tr><td>Soxhlet Apparatus</td><td>Sigma Aldrich</td><td>64826</td><td></td></tr><tr><td>Extraction Thimble</td><td>Sigma Aldrich</td><td>64842</td><td></td></tr><tr><td>B&amp;uuml;chner Funnel</td><td>Chemglass</td><td>CG-1406-25</td><td></td></tr><tr><td>High Pressure Reaction Vessel</td><td>Chemglass</td><td>CG-1880-12</td><td></td></tr><tr><td>Whatman Filter Paper</td><td>GE Life Sciences</td><td>1442-042</td><td>Grade 42, Ash 0.007%, circle, 42.5 mm</td></tr><tr><td>Biodiesel (B100)</td><td>Bellingham Shell</td><td></td><td>The biodiesel (B100) in &lt;strong&gt;Figure 3&lt;/strong&gt; was purchased at a local filling station: Bellingham Shell, Bellingham, WA 98226</td></tr><tr><td>&lt;em&gt;Isochrysis&lt;/em&gt;</td><td>Aquacave</td><td></td><td>In addition to Reed and Necton, &lt;em&gt;Isochrysis&lt;/em&gt; can also be purchased from Aquacave (Gurnee, IL) at: www.aquacave.com (accessed September 30, 2015).</td></tr><tr><td>&lt;em&gt;Isochrysis&lt;/em&gt;</td><td>Brine Shrimp Direct</td><td></td><td>&lt;em&gt;Isochrysis&lt;/em&gt; can also be purchased from Brine Shrimp Direct (Ogden, UT) at: www.brineshrimpdirect.com (accessed September 30, 2015).</td></tr></tbody></table>

experimental protocol for biodiesel production with isolation of alkenones as coproducts from commercial isochrysis algal biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Prior to processing, the Isochrysis paste (Iso-paste) was first dried. This was conveniently accomplished on larger scale by adding the Iso-paste to a large crystallizing dish and allowing the material to air-dry at room temperature. During drying, some pooled water forms (generally reddish colored) that can be removed by decanting or pipetting to accelerate the drying process. After approximately 48 - 96 hr, the now dry Isochrysis could be scraped out of the crystallizing dish and obtained as a black/green flaky material with a seaweed-like smell (Figure 2). Yields of dry biomass were generally 20% w/w of the paste as advertised. By contrast, the powdered Isochrysis product (Iso-powder) was a yellow-brown, finely milled, dry powder (95% dry) that was used directly without further processing (Figure 2).
<img alt="Figure 2" src="/files/ftp_upload/54189/54189fig2.jpg" /> 
Figure 2. Comparison of commercial Isochrysis. Isochrysis paste (80% wet) is spread along the bottom of a crystallizing dish and left to air-dry at room temperature for 48-96 hr before processing. The resulting dried Isochrysis is obtained as a dark-colored flaky material (right) that is different in appearance than the commercial dry powder Isochrysis (left). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54189/54189fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Extraction of either the dried Iso-paste or Iso-powder by Soxhlet with hexanes gave, after removal of the hexanes, algal oils (h-AO) that were similar in appearance as dark-green/near-black solids (mp. ~ 50-60 &#176;C). Yields of h-AO when starting from the paste (&#34;Iso-paste-hAO&#34;) were typically 20% w/w of the dry Isochrysis biomass, consistent with our previous results,26 whereas yields of h-AO by Soxhlet extraction of the commercial powdered Isochrysis (&#34;Iso-powder-hAO&#34;) were 15% w/w (Table 1).
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Product (g)</td>
			<td>Iso-paste</td>
			<td>Iso-powder-1</td>
			<td>Iso-powder-2</td>
		</tr>
		<tr>
			<td>Dry Biomass&#160;</td>
			<td>30</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Hexane Algal Oil&#160;</td>
			<td>5.86</td>
			<td>2.87</td>
			<td>3.11</td>
		</tr>
		<tr>
			<td>FFAs&#160;</td>
			<td>3.52</td>
			<td>1.34</td>
			<td>1.38</td>
		</tr>
		<tr>
			<td>Neutral lipids&#160;</td>
			<td>2.34</td>
			<td>1.38</td>
			<td>1.61</td>
		</tr>
		<tr>
			<td>Alkenones&#160;</td>
			<td>0.94</td>
			<td>0.63</td>
			<td>0.74</td>
		</tr>
		<tr>
			<td colspan="4">Notes: Iso-powder-1 and Iso-powder-2 represent results from two samples of powdered Isochrysis that were processed in parallel. For other reports with yields of these products from Iso-paste see references 26, 32, and 33.</td>
		</tr>
	</tbody>
</table>
Table 1. Product yields from commercial Isochrysis biomass.
Acylglycerols in the h-AO were converted to the corresponding water-soluble carboxylate salts (i.e., soaps) upon addition of aqueous KOH in methanol/CH2Cl2. Neutral lipids including alkenones were then extracted from this aqueous mixture by selective partitioning with hexanes. After removal of the neutral lipids, reacidification of the soaps then produced the corresponding free fatty acids (FFAs) that could be similarly extracted from the aqueous phase with hexanes. The overall mass recoveries for combined FFAs and neutral lipids from either Iso-paste-hAO or Iso-powder-hAO were consistently near quantitative. However, the ratio of products (i.e., neutral lipids + FFAs) was different. From the Iso-paste-hAO we obtained 60% (w/w) FFAs and 40% (w/w) neutral lipids (Table 1). Conversely, Iso-powder-hAO proved enriched in neutral lipids (average = 54% neutral lipids + 46% FFAs) as detailed in Table 1.
Esterification of the FFAs with H2SO4 and methanol then produced fatty acid methyl esters (FAMEs, i.e., biodiesel) as a dark green near black oily liquid in greater than 90% yield (Figure 3). Decolorization by heating over Montmorillonite K1029 (MK10) clay then gave a yellow/orange product, similar in appearance to other commercial biodiesel fuels (see List of Materials) (Figure 3). Results from the FAME analysis of decolorized Isochrysis biodiesel fuels are shown in Table 2.
<img alt="Figure 3" src="/files/ftp_upload/54189/54189fig3.jpg" /> 
Figure 3. Comparison of Isochrysis and soybean biodiesel fuels. Green Isochrysis biodiesel (middle) is produced by esterification of extracted and purified free fatty acids. Decolorization produces a product (right) with similar properties to commercial biodiesel (left). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54189/54189fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>FAMEA</td>
			<td>Iso-paste</td>
			<td>Iso-powder</td>
		</tr>
		<tr>
			<td>14:00</td>
			<td>16.4</td>
			<td>19.4</td>
		</tr>
		<tr>
			<td>14:01</td>
			<td>-</td>
			<td>0.3</td>
		</tr>
		<tr>
			<td>15:00</td>
			<td>trace</td>
			<td>0.3</td>
		</tr>
		<tr>
			<td>16:00</td>
			<td>10.1</td>
			<td>8.8</td>
		</tr>
		<tr>
			<td>16:1 &#916;9</td>
			<td>7.6</td>
			<td>5.5</td>
		</tr>
		<tr>
			<td>16:02</td>
			<td>ND</td>
			<td>0.3</td>
		</tr>
		<tr>
			<td>16:03</td>
			<td>ND</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>18:00</td>
			<td>Trace</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>18:1B</td>
			<td>12.1</td>
			<td>14.3</td>
		</tr>
		<tr>
			<td>18:02</td>
			<td>8.1</td>
			<td>7.1</td>
		</tr>
		<tr>
			<td>18:3C</td>
			<td>8.5</td>
			<td>13.5</td>
		</tr>
		<tr>
			<td>18:04</td>
			<td>19.8</td>
			<td>10.4</td>
		</tr>
		<tr>
			<td>18:05</td>
			<td>ND</td>
			<td>3</td>
		</tr>
		<tr>
			<td>20:05</td>
			<td>ND</td>
			<td>-</td>
		</tr>
		<tr>
			<td>22:05</td>
			<td>ND</td>
			<td>2</td>
		</tr>
		<tr>
			<td>22:06</td>
			<td>6.9</td>
			<td>11</td>
		</tr>
		<tr>
			<td>&#931;D</td>
			<td>89.8</td>
			<td>96.2</td>
		</tr>
		<tr>
			<td colspan="3">Notes: AFatty acid nomenclature is #carbons:#cis-double bonds. BCombined 18:1 &#916;9 + 18:1 &#916;11. CCombined &#916;6,9,12 and &#916;9,12,15 isomers. DThe remaining material is roughly 50:50 other FAMEs and non-FAME components (Total ~95% FAME). ND = Not detected.</td>
		</tr>
	</tbody>
</table>
Table 2. FAME composition of biodiesel produced from commercial Iso-past and Iso-powder Isochrysis biomass.
Neutral lipids were obtained as a greenish solid mixture at 40% w/w from the Iso-paste-hAO and 54% (avg.) from the Iso-powder-hAO (Table 1). Filtering the dissolved neutral lipids through silica using DCM gave after removal of the solvent, a reddish/orange solid that could be recrystallized with hexanes to afford analytically pure alkenones as a white solid. This procedure resulted in 16% (w/w) isolated yield of alkenones from Iso-paste-hAO and 25% yield from Iso-powder-hAO (Table 1).

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Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

experimental-protocol-for-biodiesel-production-with-isolation

Nanyang Technological University

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20.7K Views.  Western Washington University.  Methoden worden voor de productie van biodiesel, samen met de co-isolatie van alkenones waardevolle coproducten van commerciële Isochrysis microalgen. 

Video: Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Chemistry

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Bioengineering

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

Developmental Biology

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Biodiesel Synthesis by Ultrasonic-Assisted Transesterification of Vegetable Oils

Ultrasonic-Assisted Preparation of Biodiesel Products from Vegetable Oils

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel synthesis. This alkaline-catalyzed procedure harnesses ultrasound as a potent energy input, facilitating the rapid conversion of extra virgin olive oil into biodiesel. In this demonstration, the reaction is run in an ultrasonic bath under ambient conditions for 15 min, requiring a 1:6 molar ratio of extra virgin olive oil to methanol and a minimum amount of KOH as the catalyst. The physiochemical properties of biodiesel are also reported. Emphasizing the remarkable advantages of ultrasonic-assisted transesterification, this method demonstrates notable reductions in reaction and separation times, achieving near-perfect purity (~100%), high yields, and negligible waste generation. Importantly, these benefits are achieved within a framework that prioritizes safety and environmental sustainability. These compelling findings underscore the effectiveness of this approach in converting vegetable oil into biodiesel, positioning it as a viable option for both research and practical applications.

Author Spotlight: Employing Green-Chemistry Principles for Safe and Sustainable Synthesis of Biodiesels

A safe ultrasonic-assisted transesterification method for vegetable oils using an alkaline catalyst is presented here. The method is rapid and efficient for preparing pure biodiesel products.

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

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