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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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/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 border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Isochrysis</td>
			<td style="width:107px;">Reed Mariculture</td>
			<td style="width:119px;">Iso, Raw, Unprocessed, 20%dw</td>
			<td style="width:167px;">Live culture paste, 20% w/w biomass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isochrysis</td>
			<td style="width:107px;">Necton, S.A</td>
			<td style="width:119px;">AADISS004</td>
			<td>PhytoBloom Prof Isochrysis (Freeze-dried)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hexanes</td>
			<td style="width:107px;">Fisher Chemical</td>
			<td style="width:119px;">H291-4</td>
			<td>ACS Certified</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methanol</td>
			<td style="width:107px;">Fisher Chemical</td>
			<td style="width:119px;">A452-4</td>
			<td>HPLC Grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dichloromethane</td>
			<td style="width:107px;">Fisher Chemical</td>
			<td style="width:119px;">D37-4</td>
			<td>Certified/Stabilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soxhlet Apparatus</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">64826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Extraction Thimble</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">64842</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B&#252;chner Funnel</td>
			<td style="width:107px;">Chemglass</td>
			<td style="width:119px;">CG-1406-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High Pressure Reaction Vessel</td>
			<td style="width:107px;">Chemglass</td>
			<td style="width:119px;">CG-1880-12</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Whatman Filter Paper</td>
			<td style="width:107px;">GE Life Sciences</td>
			<td>1442-042</td>
			<td>Grade 42, Ash 0.007%, circle, 42.5 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biodiesel (B100)</td>
			<td style="width:107px;">Bellingham Shell</td>
			<td style="width:119px;"></td>
			<td>The biodiesel (B100) in Figure 3 was purchased at a local filling station: Bellingham Shell, Bellingham, WA 98226</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isochrysis</td>
			<td style="width:107px;">&#160;Aquacave</td>
			<td style="width:119px;"></td>
			<td>&#160;In addition to Reed and Necton, Isochrysis can also be purchased from (Aquacave. (Gurnee, IL) at: www.aquacave.com (accessed September 30, 2015).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isochrysis</td>
			<td style="width:107px;">Brine Shrimp Direct</td>
			<td style="width:119px;"></td>
			<td>Isochrysis 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/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/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).

Watch this Scientific Journal Video about Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass at JoVE.com

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

Research

JoVE Journal

Environment

20.6K Views.  Western Washington University. Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

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

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted to better equalize conditions occurring at both scales. During the short 8-week representative time period, starting ammonia and total nitrogen concentrations ranged from 3.1-14 mg NH3-N/L, and 8.1-20.1 mg N/L, respectively. The performance of the treatment system was evaluated based on its ability to remove ammonia and total nitrogen and to produce algal biomass. Mean &#177; standard deviation of ammonia removal, total nitrogen removal and biomass growth rates were 0.95&#177;0.3 mg NH3-N/L/day, 0.89&#177;0.3 mg N/L/day, and 0.02&#177;0.03 g biomass/L/day, respectively. All vessels showed a positive relationship between the initial ammonia concentration and ammonia removal rate (R2=0.76). Comparison of process efficiencies and production values measured in reactors of different scale may be useful in determining if lab-scale experimental data is appropriate for prediction of commercial-scale production values.

An experimental methodology is presented to compare the performance of small (100 L) and large (1,000 L) scale reactors designed for algae remediation of landfill wastewater. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted based on application.

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

Experimental System of Solar Adsorption Refrigeration with Concentrated Collector

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The main components of the system were the adsorbent bed, the condenser, the evaporator, the cooling sub-system, and the solar collector. In the first step of the experiment, the vapor-saturated bed was heated by the solar radiation under closed conditions, which caused the bed temperature and pressure to increase. When the bed pressure became high enough, the bed was switched to connect to the condenser, thus water vapor flowed continually from the bed to the condenser to be liquefied. Next, the bed needed to cool down after the desorption. In the solar-shielded condition, achieved by aluminum foil, the circulating water loop was opened to the bed. With the water continually circulating in the bed, the stored heat in the bed was took out and the bed pressure decreased accordingly. When the bed pressure dropped below the saturation pressure at the evaporation temperature, the valve to the evaporator was opened. A mass of water vapor rushed into the bed and was adsorbed by the zeolite material. With the massive vaporization of the water in the evaporator, the refrigeration effect was generated finally. The experimental result has revealed that both the COP (coefficient of the performance of the system) and the SCP (specific cooling power of the system) of the SAPO-34 zeolite was greater than that of the ZSM-5 zeolite, no matter whether the adsorption time was longer or shorter. The system of the SAPO-34 zeolite generated a maximum COP of 0.169.

With solar energy as the driving force, a novel adsorption refrigeration system has been developed and experimentally investigated. Water vapor and zeolite formed the working pair of the adsorption system. This manuscript describes the setup of the experimental rig, the operation procedure, and the important results.

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...