The authors would like to acknowledge the technical and financial support provided by the Agricultural Experiment Station at the University of Georgia. In addition, the authors want to thank Samuel Ogundipe, Dr. Ronald Pegg, and Dr. Joon Hyuk Suh for their help in analyzing process samples.

The reagents, consumables, and equipment used in this study are listed in the Table of Materials.
1. Constructing and integrating the bioreactor and the liquid-liquid extraction system
<ol>
	<li>Prepare the organic-phase extraction solution and reservoir.
	<ol>
		<li>Prepare 2 L of organic-phase extraction solution by dissolving 60 g of trioctylphosphine oxide (TOPO) in mineral oil using a magnetic stir plate and a stirrer.</li>
		<li>Add the extraction solution to a 2&#160;L glass reservoir (i.e., Schott bottle).</li>
		<li>Place the reservoir on a magnetic stir plate (Figure 1A). The recommended mixing speed during continuous LLE operation is 150-250 rpm.</li>
	</ol>
	</li>
	<li>Prepare a three-port cap for the extraction solution reservoir.
	<ol>
		<li>Attach a dip tube to the first port to serve as an out-flow port supplying extraction solution to the forward extraction membrane (FEM) (Figure 1B).</li>
		<li>Affix a second port to serve as a return port for the extraction solution from the backward extraction membrane (BEM) (Figure 1C).</li>
		<li>Add a third port, open to the atmosphere, to dampen pressure fluctuations caused by the diaphragm pump.</li>
	</ol>
	</li>
	<li>Connect the diaphragm pump to the extraction membranes.
	<ol>
		<li>Place a 100 rpm variable speed pump drive equipped with a polytetrafluoroethylene (PTFE) diaphragm pump head adjacent to the reservoir (Figure 1D).</li>
		<li>Connect the extraction solution reservoir out-flow port (Figure 1A) to the inlet of the diaphragm pump and then the outlet of the diaphragm pump to the shell-side inlet at the base of the FEM (Figure 1B) using the flexible pump tubing (e.g., size 16, 18).</li>
		<li>Connect the shell-side outlet at the top of the FEM to the shell-side inlet at the base of the BEM (Figure 1C) using flexible pump tubing (e.g., size 18).</li>
		<li>Connect the shell-side outlet at the top of the BEM to the return port on the extraction solution reservoir (Figure 1A) using flexible pump tubing (e.g., size 16, 18). 
		NOTE:&#160;This system component transfers MCFAs extracted from the fermentation broth in the FEM to the stripping solution in the BEM.</li>
	</ol>
	</li>
	<li>Prepare the aqueous-phase stripping solution and reservoir.
	<ol>
		<li>Prepare 3.25&#160;L of aqueous-phase stripping solution by taking a 0.5 M boric acid solution and adjusting it to pH 9 using NaOH.</li>
		<li>Pour the solution into a 3.5&#160;L glass reservoir with a magnetic stir bar.</li>
		<li>Place the reservoir on a magnetic stir plate (Figure 1E).</li>
	</ol>
	</li>
	<li>Prepare a four-port cap for the stripping solution reservoir.
	<ol>
		<li>Attach a dip tube to the first port to serve as an out-flow port supplying stripping solution to the BEM.</li>
		<li>Affix a second port comprising a Y-fitting to (1) serve as the return-flow port of the stripping solution from the BEM and (2) serve as an entrainment stream for NaOH additions from the pH control system.</li>
		<li>Add a third port, open to the atmosphere, to account for the increase in volume caused by NaOH additions and MCFA accumulation.</li>
		<li>Provide a fourth port to accommodate the pH probe of the pH controller.</li>
	</ol>
	</li>
	<li>Install a pH-control system at the stripping solution reservoir.
	<ol>
		<li>Integrate a pH-control system with a set point of pH 9 with the stripping solution reservoir (Figure 1F). Use 5 M NaOH as a base solution with the pH controller to counteract the collected MCFAs.</li>
		<li>Insert the pH probe through the stripping solution reservoir port and suspend it in the stripping solution. 
		NOTE: This pH-control system does not require an acid solution.</li>
	</ol>
	</li>
	<li>Prepare the bioreactor connection ports.
	<ol>
		<li>Designate two ports, an out-flow port and a return-flow port, at the bioreactor for connection with the liquid-liquid extraction (LLE) system (Figure 1G,H).</li>
		<li>Connect a dip tube to the out-flow port. The MCFA-rich broth will be pumped from the bioreactor at the out-flow port into the LLE system. 
		NOTE: The broth first enters the hollow-fiber membrane filter before the extraction membranes to remove the cells and other solids to prevent fouling.</li>
		<li>Insert a tee-fitting at the return-flow port to receive: (1) MCFA-depleted broth recycled from the FEM and (2) cell-containing retentate from the hollow-fiber membrane filter.</li>
	</ol>
	</li>
	<li>Install the hollow-fiber membrane filter.
	<ol>
		<li>Place a 300 rpm variable-speed pump drive adjacent to the bioreactor (Figure 1I).</li>
		<li>Affix the hydrophilic hollow-fiber membrane (Figure 1J) to a ring stand above the peristaltic pump.</li>
		<li>Stack two peristaltic pump heads on the pump drive, large (e.g.,&#160;size 17) and small (e.g., size 16). 
		&#8203;NOTE: The larger pump head is connected to the hollow-fiber membrane filter to ensure the permeate flow rate is greater than the flow rate feeding into the FEM. If this were not the case, the permeate would be consumed faster than it is produced, causing a vacuum to form.</li>
		<li>Connect the out-flow port at the bioreactor to the large pump head inlet using flexible pump tubing (e.g.,&#160;size 17).</li>
		<li>Connect the large pump head outlet to the hydrophilic hollow-fiber membrane filter at the tube-side inlet at the base of the filter using flexible pump tubing (e.g.,&#160;size 17).</li>
		<li>Cap the top shell-side port of the filter to prevent the influx of air. 
		NOTE:The MCFA-rich broth (containing cells) will flow up through the hollow fiber tubes and return to the bioreactor. Clear broth (cell-free) will pass through the 0.2 &#181;m hydrophilic polyethersulfone (PES) membrane and collect on the shell side of the filter.</li>
	</ol>
	</li>
	<li>Connect the FEM.
	<ol>
		<li>Affix the first hydrophobic hollow-fiber membrane module (FEM) to a ring stand above the pump (Figure 1B).</li>
		<li>Connect the hydrophilic hollow-fiber membrane filter (Figure 1J) at the shell-side outlet to the small pump head inlet using flexible pump tubing (e.g., size 16, 18).</li>
		<li>Connect the small pump head outlet to the FEM at the tube-side inlet at the base of the module using flexible pump tubing (e.g.,&#160;size 16, 18).</li>
		<li>Connect the pressure gauge (Figure 1K) and flow-restriction valve (Figure 1L) to the tube-side outlet of the FEM using a coupling fitting and tee-fitting.</li>
		<li>Connect the tube-side outlet of the FEM to the return port at the bioreactor using flexible pump tubing (e.g.,&#160;size 18). 
		&#8203;NOTE:This system component transfers the MCFAs from the clear fermentation broth to the extraction solution and then returns the clear broth to the bioreactor.</li>
	</ol>
	</li>
	<li>Connect the BEM.
	<ol>
		<li>Place a 300 rpm variable speed pump drive equipped with a peristaltic pump head (e.g., size 16)&#160;adjacent to the bioreactor (Figure 1M).</li>
		<li>Affix the second hydrophobic hollow-fiber membrane module to a ring stand above the peristaltic pump (Figure 1C).</li>
		<li>Connect the stripping solution reservoir out-flow port to the peristaltic pump inlet and the pump outlet to the tube-side inlet at the base of the BEM using flexible pump tubing (e.g.,&#160;size 16, 18).</li>
		<li>Connect the pressure gauge and flow-restriction valve to the tube-side outlet of the BEM using a coupling fitting and tee-fitting.</li>
		<li>Connect the tube-side outlet at the top of the BEM to the stripping solution reservoir return-flow port using flexible pump tubing (e.g.,&#160;size 16, 18). 
		NOTE:&#160;This system component transfers the MCFAs from the extraction solution to the stripping solution in the BEM and then returns the stripping solution to its reservoir.</li>
	</ol>
	</li>
</ol>
2. Initiatiing the operation of the liquid-liquid extraction system
<ol>
	<li>Prime and circulate the aqueous phase lines.
	<ol>
		<li>Turn on the stripping solution peristaltic pump/BEM (Figure 1M) and set the pump speed to achieve a constant flow rate between 25-250 mL&#183;min-1. The BEM can accommodate a relatively large range of flow rates. Start with a conservatively high flow rate initially to ensure sufficient MCFA extraction. The flow rate can be incrementally reduced later during operation to extend the service life of the pump equipment and tubing. 
		NOTE: The flow speed should not be so low as to allow an accumulation of MCFAs in the bioreactor broth (see Representative Results).</li>
		<li>Slowly close the needle valve at the shell-side outlet of the BEM (Figure 1C) to establish a back-pressure of ~5 psig.</li>
		<li>Turn on the bioreactor/FEM peristaltic pump (Figure 1I) and set the pump speed to achieve a constant flow rate between 25-250 mL&#183;min-1. The FEM can accommodate a relatively large range of flow rates. Start with a conservatively high flow rate initially to ensure sufficient MCFA extraction. The flow rate can be incrementally reduced later during operation to extend the service life of the pump equipment and tubing. 
		NOTE: The flow speed should not be so low as to allow an accumulation of MCFAs in the bioreactor broth (see Representative Results section).</li>
		<li>Slowly close the needle valve at the shell-side outlet of the FEM (Figure 1B) to establish a back-pressure of ~5 psig.</li>
		<li>Visually check the return-flow lines to ensure a constant flow and that the lines have been primed.</li>
		<li>Verify that clear broth is collected within the shell side of the hollow-fiber membrane filter (Figure 1J). 
		NOTE: It will take several hours to fill the FEM and establish a steady flow between the hollow-fiber membrane filter and the FEM. The flow speed should not be so low as to allow an accumulation of MCFAs in the bioreactor broth (see Representative Results section).</li>
	</ol>
	</li>
	<li>Prime and circulate the organic phase lines.
	<ol>
		<li>Turn on the organic-phase extraction solution diaphragm pump (Figure 1D) and set the pump speed to achieve a constant flow rate between 5.0-50 mL&#183;min-1. Start with a conservatively low flow rate initially to minimize the pressure in the organic-phase extraction and minimize the risk of cross-over. If necessary, the flow rate can be incrementally increased later during operation to improve extraction efficiency</li>
		<li>Wait until the FEM and BEM are filled.</li>
		<li>Visually check the return-flow port at the extraction solution reservoir to ensure constant flow.</li>
		<li>Verify that no organic phase solution is crossing over into the stripping solution or fermentation broth lines. If a cross-over is occurring, small droplets of the organic phase can be seen. If this happens, decrease the diaphragm pump speed and increase the back pressure slightly at the FEM or BEM as appropriate. Do not exceed 10 psig. 
		NOTE:&#160;To prevent membrane cross-over, it is important to establish flow and back-pressure within the aqueous phase lines before priming the organic phase lines.</li>
	</ol>
	</li>
	<li>Continuously extract MCFAs from the bioreactor.
	<ol>
		<li>The LLE system should be fully operational. Allow the system to run continuously during bioreactor operation.</li>
		<li>Measure MCFA concentrations in the bioreactor daily to ensure sufficient extraction of MCFAs. If elevated MCFA concentrations in the bioreactor occur, that usually indicates insufficient flow rates of the fermentation broth through the FEM. It may also indicate diminished membrane flux due to fouling and the need for maintenance (see step 2.6). 
		NOTE: SCFA and MCFA concentrations can be measured via gas chromatography according to the method described by Ge et al.11.</li>
	</ol>
	</li>
	<li>Monitor MCFA accumulation in the stripping solution reservoir.
	<ol>
		<li>Measure the MCFA concentration within the stripping solution daily during the batch cycle. The LLE system continuously transfers MFCAs from the bioreactor to the stripping solution, increasing the MCFA concentration over time. The process can run for extended periods to produce high MCFA titers. The volumetric production rate of MCFAs (mM C&#183;L-1&#183;d-1) can be estimated using Equation 111. 
		NOTE: Volumetric Production Rate = <img alt="Equation 1" src="/files/ftp_upload/66956/66956eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(Eq. 1) 
		where: 
		Cb,n= MCFA concentration in the fermentation broth on Day n, mM C 
		Cs,n= MCFA concentration in the stripping solution on Day n, mM C 
		Cs,n-1= MCFA concentration in the stripping solution on Day n-1, mM C 
		Vs,n= Volume of stripping solution on Day n, L 
		Vb,n= Volume of fermentation broth (bioreactor volume) on Day n, L 
		HRTn= Hydraulic retention time of bioreactor on Day n, d 
		Tn= Day n, d 
		Tn-1= Day n-1, d</li>
		<li>To ensure stable operation, periodically calculate the extraction rate (mM C&#183;d-1) of the LLE system by measuring the change in MCFA concentration between measurement time points and applying Equation 211. 
		NOTE: <img alt="Equation 2" src="/files/ftp_upload/66956/66956eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;(Eq. 2) 
		where: 
		Cs,n= MCFA concentration in the stripping solution on Day n, mM C 
		Cs,n-1= MCFA concentration in the stripping solution on Day n-1, mM C 
		Vs,n= Volume of stripping solution on Day n, L 
		Tn= Day n, d 
		Tn-1= Day n-1, d</li>
		<li>To maintain adequate transfer rates from the extraction solution, replace the stripping solution with a fresh batch before the MCFA concentration reaches 80% saturation. 
		NOTE:&#160;The maximum solubility of n-caproic acid is 10.3 g&#183;L-1 at 25 &#176;C, and that of n-caprylic acid is 0.67 g&#183;L-1 at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Replace the stripping solution.
	<ol>
		<li>Turn off the diaphragm pump (Figure 1D).</li>
		<li>Turn off the stripping solution peristaltic pump (Figure 1M).</li>
		<li>Use hose clamps to clamp the tube-side inlet and the tube-side outlet of the BEM.</li>
		<li>Turn off the pH control system and remove the stripping solution reservoir cap while keeping the port connections attached (if possible).</li>
		<li>Take away the stripping solution reservoir (Figure 1E).</li>
		<li>Replace the stripping solution reservoir with a fresh batch of aqueous 0.5 M boric acid solution adjusted to pH 9 using NaOH (see step 1.4). Re-affix the cap to the reservoir.</li>
		<li>Remove the hose clamps from the tube-side inlet and the tube-side outlet of the BEM.</li>
		<li>Turn on the stripping solution peristaltic pump (Figure 1M), followed by the diaphragm pump (Figure 1D). System operation is now restored.</li>
	</ol>
	</li>
	<li>Membrane maintenance.
	<ol>
		<li>Remove the FEM and BEM from the LLE system once every three months for cleaning. Three months is a conservative estimated cleaning frequency. 
		NOTE: Depending on the application, users may clean the membranes more or less frequently. Signs of diminished membrane performance are described in the Representative Results section. During maintenance, the pumps and pH controller should be turned off. Cleaning instructions should be provided by the membrane manufacturer.</li>
		<li>Drain the liquids from the LLE system into separate containers, starting with the organic-phase extraction solution lines, followed by the fermentation broth lines, and the stripping solution lines.</li>
		<li>Once the membranes are cleaned and re-installed, return the liquids to their respective reservoirs.</li>
		<li>Re-initiate the LLE system using the approach described above (see&#160;steps 2.1-2.2).</li>
	</ol>
	</li>
</ol>

Sustainable MCFA Production via Chain Elongation and Liquid-Liquid Extraction

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There is no conflict of interest.

Biologically-produced MCFAs are commonly found in mixtures alongside various organic compounds, including SCFAs and alcohols2. Consequently, a selective separation process is necessary to recover and utilize them effectively. The LLE system developed here selectively extracts MCFAs from these mixtures continuously while conserving SCFAs and alcohols. This functionality makes the LLE system particularly suitable for fermentation applications, such as microbial chain elongation, where MCFAs, SCFAs, and alcohols constitute the primary metabolites8. Specifically, the LLE system allows the chain-elongation process to proceed by removing MCFAs, preventing product inhibition1, while leaving the SCFA and alcohol reactants in the fermentation broth for subsequent biological conversion. The LLE system can be customized for other applications by modifying the specific extraction solution. For example, continuous extraction of SCFAs produced during&#160;fermentation could be achieved using the same LLE system by removing TOPO from the extractant solution mixture.
Hence, the significance of the LLE method lies in providing a more robust MCFA extraction technique for these bioprocessing and biotechnology applications compared to other methods. In situ biphasic extraction with non-miscible liquids is another approach to extracting MCFAs from fermentation broth15. However, this approach is relatively inefficient. Emulsion layers form between the aqueous phase (i.e.,&#160;fermentation broth) and the organic phase, severely limiting mass transfer rates. Minimal interfacial fluid mixing between the phase layers also limits mass transfer. Another disadvantage is that microbial cells are in direct contact with the organic phase, causing entrainment, inhibition, and cell death15. Finally, in situ biphasic extraction requires frequent maintenance to remove and replace the organic phase.
Applying high dilution rates within the bioreactor is another method to avoid product inhibition16. High dilution rates can achieve high productivity by maintaining high reactant concentrations in the bioreactor. However, this approach is disadvantageous because it contributes to biomass washout, the generation of large effluent volumes, and high substrate losses (i.e., SCFAs and alcohols), resulting in low yields. These disadvantages can be mitigated using immobilized biomass and effluent recycling, but these interventions add to system complexity17. Finally, the MCFA concentration in the product stream is dilute, making MCFA inefficient and costly.
A new extraction approach could involve continuously distilling the MCFAs with a single forward extraction membrane that physically separates the organic and aqueous phases, thereby retaining and protecting the microbial biomass. The MCFAs would be selectively extracted into the organic phase and then distilled. The raffinate could be continuously recycled to the extraction membrane. Continuous distillation, however, is technically challenging, especially in laboratory settings, and may cause the deterioration or loss of the chemical extractant during long-term operation. Distillation may also cause thermal degradation of the organic phase and MCFA products18.
The LLE process avoids many of the disadvantages associated with these alternative approaches by incorporating several critical features and processing steps. First, the hydrophilic hollow-fiber membrane filter serves the dual purpose of protecting biomass cells (the biocatalysts) from exposure to the extractant solution in the FEB while providing a clear MCFA-rich filtrate that reduces fouling and solid accumulation in the LLE system. Second, to prevent liquid cross-over, we incorporated needle valves to create back pressure on the tube side of each membrane contactor. This precaution maintains a slight transmembrane pressure gradient, preventing undesirable leakage of the hydrophobic organic solvent from the shell side to the aqueous tube side in the FEM and BEM. In addition, the liquid streams are configured to flow in parallel from the base to the top of the FEM and BEM to prevent the entrapment of gas bubbles that could collect inside the membrane modules, reducing the transfer efficiency and causing carry-over. Furthermore, this method uses a diaphragm pump with a chemically resistant PTFE pump head to pump the corrosive MCFA-containing extractant solution, safeguarding the system from corrosion and breakdowns that could compromise the extraction process. Finally, the pH-controlled alkaline stripping solution maintains a pH gradient that allows the continuous transfer of MCFAs through the LLE system at high rates from the bioreactor to the stripping solution reservoir, where the MCFAs deprotonate and accumulate to high titers, facilitating downstream product recovery.
This LLE method is appropriate for continuous MCFA extraction from laboratory-scale bioreactors (up to a 6 L working volume) and has been validated for long-term operation in several studies1,9,11,19. The LLE method can also be applied for larger-scale applications14 (i.e., pilot-scale bioreactors) but requires proportionally scaled membranes and fluid-handling equipment. However, the method does have some limitations, mainly in the area of maintenance and system complexity. Because the process is designed to operate continuously, the membrane modules and pumps must be serviced frequently, resulting in considerable downtimes. Another drawback is that the stripping solution requires relatively large amounts of NaOH and boric acid. Moreover, MCFAs are corrosive and cause certain LLE system components to deteriorate over time. For instance, plastic connectors and the membrane housing may become brittle, requiring replacement during operation. Finally, the fluid handling network in the LLE system is complex, involving many connection points that are liable to develop leaks. Most of these limitations and drawbacks, however, are typical of continuous membrane separation processes and should be expected.
Overall, this LLE protocol offers a robust and efficient approach for selective MCFA extraction, which has implications for advancing research in diverse fields. The method could find many relevant applications in the field of precision fermentation for in-situ recovery of extracellular metabolite products during fermentation. LLE could be a lower-cost alternative to conventional downstream processing (DSP) approaches, such as post-run centrifugation, micro- and ultra-filtration, or solvent extractions performed in batches. Indeed, DSP often represents a major cost driver in industrial fermentation processes. Continuous product extraction using LLE may also enable continuous fermentations, dramatically improving the operations&#39; productivity and run-time efficiencies compared to conventional batch or fed-batch approaches. Also, future research could investigate extractant mediums other than organic solvents, such as deep eutectic solvents or ionic liquids. Lastly, the LLE system described in this protocol was intended for experimental purposes in a laboratory setting; thus, there is still considerable room for optimization studies to reduce energy requirements, membrane area, and overall extraction yields and rates.

Medium-chain fatty acids (MCFAs) are high-value building block chemicals comprising chain lengths ranging from six (C6) to twelve (C12) carbons. MCFAs have industrial applications in foods, animal feeds, pharmaceuticals, cosmetics, fragrances, antimicrobial agents, and chemical synthesis1,2,3. Currently, most MCFAs derive from palm and coconut oil sourced from Southeast Asia and South America4,5. The severe ecological damage associated with palm and coconut oil production is well-recognized by stakeholders and the general public. Researchers are exploring biological approaches (e.g., precision and open-culture fermentations) to generate MCFAs more sustainably using low-value substrates or organic wastes as feedstock6,7. One sustainable way to produce MCFAs is by upcycling organic waste streams using a process called microbial chain elongation (CE). This secondary fermentation bioprocess is similar to anaerobic digestion in that it exploits the versatility of anaerobic open-culture microbiomes, but instead of promoting methane formation, CE systems deliberately suppress the methanogenic pathway. In a microbiome where carbon cannot be maximally reduced to CH4, nor H2 maintained below 10-4 atm by hydrogen-consuming archaea, the &#946;-oxidation reaction that would normally break down longer chain carboxylates to acetate (e.g., C6 &#8594; C4 &#8594; C2) can be reversed (e.g., C2 &#8594; C4 &#8594; C6, etc.), so long as a reduced compound (i.e., electron donor) such as ethanol or lactate is supplied8. In this metabolism, the fatty acid molecule undergoing elongation serves as the electron acceptor. Thus, instead of generating a product with a carbon length of one (CH4) as in anaerobic digestion, the CE process generates MCFAs with carbon lengths ranging from six to eight. A large and growing market is ready to receive these green platform chemicals. However, thus far, the CE process has not been shown to produce MCFAs with carbon lengths exceeding eight carbons at appreciable rates.
Efficient extraction of these MCFAs is important not only for the recovery of the desired product but also to prevent product inhibition and push the microbiome toward producing more MCFAs1. As the concentration of MCFAs increases, MCFA metabolism is inhibited and becomes less thermodynamically favorable. By removing the MCFAs continuously, production rates are maintained. Also, because SCFAs serve as the substructures for the chain-elongation process, they should not be removed from the fermentation broth. Targeted extractant mixtures should selectively extract MCFA products from fermentation broths containing SCFAs.
Here, a robust and practical approach is demonstrated to continuously extract MCFAs from SCFA-containing fermentation broth using a liquid-liquid extraction (LLE) system comprising a hydrophobic, polypropylene forward hollow-fiber membrane extractor, a selective organic extractant solution (trioctylphosphine oxide [TOPO]9,10,11), and a backward hollow-fiber membrane extractor. A cell-guard filter upstream of the LLE system is installed to retain biomass and mitigate membrane fouling. The MCFAs are forward extracted, in their protonated form, from the aqueous fermentation broth (typically with a pH set point &#60;5.8) into an organic extractant solution (i.e., 3% TOPO (w/v) in mineral oil) and then backward extracted into an alkaline stripping solution (pH = 9), where they deprotonate and concentrate to high titers for downstream processing. The particular pH set points are essential because they dictate the concentration gradient between each phase of the LLE process, ensuring a net transfer of MCFAs from the fermentation broth to the stripping solution. LLE using forward and backward extraction membranes achieve high extraction rates while minimizing alcohols and SCFAs co-extraction. The organic solvent adjuvant, TOPO, enables the formation of MCFA complexes. These complexes are more soluble in organic phases than water, resulting in high MCFA selectivity. The LLE process also avoids the many disadvantages associated with existing approaches, which will be discussed in the Discussion section. Long-term implementation using this LLE approach has been demonstrated in multiple studies9,10,11. While this approach is particularly suitable for applications involving MCFA production via microbial chain elongation, it is also useful in other applications that require selective separation of compounds possessing similar chemical properties because the organic extractant system can be customized.

<table><tbody><tr><td>10 L Media Bottle</td><td>Duran</td><td>218018658</td><td></td></tr><tr><td>3.5 L Media Bottle</td><td>Duran</td><td>218016957</td><td></td></tr><tr><td>Boric acid, 99.5%,&amp;nbsp;</td><td>ThermoScientific (Fisher Scientific)</td><td>327132500</td><td></td></tr><tr><td>Hydrophilic MINIKROS 20CM 0.2UM PES 1MM 1.5TC X 3/4TC</td><td>Repligen</td><td>N02-P20U-10-N</td><td></td></tr><tr><td>L/S Variable-Speed Pump Drive; 100 rpm</td><td>MasterFlex (VWR)</td><td>MFLX07528-10</td><td></td></tr><tr><td>L/S Variable-Speed Pump Drive; 300 rpm</td><td>MasterFlex (VWR)</td><td>MFLX07528-20</td><td></td></tr><tr><td>Light Mineral Oil, NF (4 Liters) (CAS: 8042-47-5)</td><td>Thomas Scientific</td><td>C761Z18</td><td></td></tr><tr><td>Liqui-Cel 2.5x8 X50 membrane CO2, PP Housing Viton O-rings (0.5-3 gpm (0.1-0.7 m3/h)), 1/4-in FNPT connections</td><td>3M</td><td>LC-02508X50-G453</td><td></td></tr><tr><td>Magnetic Stirrer, 20 L Capacity, 110 V</td><td>Cole-Parmer</td><td>EW-04661-29</td><td></td></tr><tr><td>Masterflex L/S Precision Pump Tubing, Tygon, Size 14</td><td>MasterFlex (VWR)</td><td>MFLX06402-14</td><td>Specific tubing size will depend on application.</td></tr><tr><td>Masterflex L/S Precision Pump Tubing, Tygon, Size 16</td><td>MasterFlex (VWR)</td><td>MFLX06402-16</td><td>Specific tubing size will depend on application.</td></tr><tr><td>Masterflex L/S Precision Pump Tubing, Tygon, Size 17</td><td>MasterFlex (VWR)</td><td>MFLX06402-17</td><td>Specific tubing size will depend on application.</td></tr><tr><td>Masterflex L/S Precision Pump Tubing, Tygon, Size 18</td><td>MasterFlex (VWR)</td><td>MFLX06402-18</td><td>Specific tubing size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 14, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07014-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 14, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07014-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 16, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07016-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 17, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07017-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 18, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07018-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex PTFE-diaphragm pump head, 10 to 100 mL/min</td><td>MasterFlex (VWR)</td><td>MFLX07090-62</td><td></td></tr><tr><td>Oakton 220 pH/ORP/Temperature Controller, 1/8 DIN</td><td>Spectrum Laboratory Products</td><td>664-12595-E1</td><td></td></tr><tr><td>Oakton 220 pH/ORP/Temperature Controller, 1/8 DIN</td><td>Spectrum Laboratory Products</td><td>664-12595-E1</td><td></td></tr><tr><td>Oakton Female BNC-to-Stripped Wire Adapter</td><td>Spectrum Laboratory Products</td><td>664-12592-E1</td><td></td></tr><tr><td>pH Probe with BNC Connector</td><td>ThermoScientific</td><td>10010-788</td><td>Any pH probe with a BNC connector will suffice.&amp;nbsp;</td></tr><tr><td>Precision Flow-Adjustment Valve, White Polypropylene, 1/4 NPT Male x Male</td><td>McMaster-Carr</td><td>7792K57</td><td></td></tr><tr><td>ProConnex&amp;nbsp; Fittings Kits - A</td><td>Repligen</td><td>ACPX-KT2-01N</td><td>Compatible with Hydrophilic MINIKROS Filter</td></tr><tr><td>ProConnex Fittings Kits - B</td><td>Repligen</td><td>ACPX-KT1-01N</td><td>Compatible with Hydrophilic MINIKROS Filter</td></tr><tr><td>Sodium Hydroxide Pellets for Analysis</td><td>Sigma Aldrich</td><td>1.06498</td><td></td></tr><tr><td>Stainless-Steel Pressure Gauge 0-60 psi Stainless Steel 1/4&quot; NPT 2.5&quot; Face Dial</td><td>NA</td><td>XJ-219</td><td>Any comparable pressure gauge covering 0-60 psig range will suffice.&amp;nbsp;</td></tr><tr><td>Trioctylphosphine oxide (TOPO)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>346187-100G</td><td></td></tr></tbody></table>

continuous liquid-liquid extraction of medium-chain fatty acids from fermentation broth using hollow-fiber membranes

Medium-chain fatty acids (MCFAs; carbon-lengths: C6-C12) are high-value platform chemicals that serve a variety of industrial applications, including green antimicrobials, food ingredients, animal feed additives, cosmetics, fragrances, pharmaceuticals, and structured lipids. Currently, most MCFAs are produced from palm and coconut oil originating from Southeast Asia and South America. The conventional approach to harvesting palm and coconut fruits causes considerable ecological damage in these regions. Therefore, researchers are developing biological approaches (e.g., precision and open-culture fermentations) to generate MCFAs more sustainably using low-value substrates (e.g., methanol, ethanol, lactate) or organic wastes as feedstock. Microbial chain-elongation (CE) is a rapidly maturing open-culture fermentation platform that converts short-chain fatty acids (SCFAs; carbon lengths: C1-C5) into a subset of these MCFAs at industrially relevant rates. However, continuous in situ extraction of MCFA products is necessary not only to avoid product inhibition but also to facilitate the recovery of MCFAs in a pure and usable form.&#160;Liquid-liquid extraction (LLE) using hollow-fiber membranes and targeted extractant mixtures has proven a robust approach to selectively extract MCFA products from fermentation broths containing SCFAs. Here, the application of LLE for continuous MCFA removal is demonstrated using CE as the reference fermentation system and 3% (w/v) trioctylphosphine oxide in mineral oil as the extractant system. Fatty acids ranging from valeric acid (C5) to caprylic acid (C8) are selectively removed from SCFA-containing broths and concentrated to high titers in a semi-batch alkaline stripping solution for downstream processing.

Positive MCFA extraction outcomes are indicated by a steady accumulation of MCFA products in the alkaline aqueous-phase stripping solution (Figure 2) and relatively stable MCFA concentrations in the fermentation broth (data not shown). Figure 2 illustrates three semi-batch cycles of the stripping solution during continuous LLE operation. A cycle comprises two stages: the batch-replacement stage (Figure 2: Day 24, Day 46, and Day 68) and the MCFA accumulation stage (Figure 2: Days 0-24, Days 25-46, Days 47-68). For this particular fermentation and LLE system, the cycle duration was approximately 20-24 days. The cycle duration will vary between applications, however, as it depends on multiple factors, including bioreactor volume, biological productivity, stripping solution volume, hollow-fiber membrane area, and liquid recirculation rates within the LLE system. During a batch cycle, the stripping solution may change color from clear to yellowish-brown due to low-level co-extraction of various small organic acids (e.g.,&#160;humic acid, fulvic acids) present in the fermentation broth (Figure 3). Negative MCFA extraction outcomes are indicated by a slow accumulation of MCFA products in the stripping solution and elevated MCFA concentrations in the fermentation broth relative to the pre-established baseline.
The biological productivity of caproate is generally higher than caprylate during these fermentation processes; therefore, it is common for caproate to accumulate at a faster rate in the stripping solution compared to caprylate. Also, it is normal for SCFAs, such as acetate and butyrate, to collect in the stripping solution in lower amounts, as seen in Figure 2. The TOPO in the mineral oil has a higher affinity for MCFAs than for SCFAs, which causes selective removal of the MCFAs. Studies by Saboe et al.12, Kaur et al.13, Carvajal-Arroyo et al.14, and Ge et al.11, demonstrated TOPO&#39;s high selectivity for fatty acids in multiple applications involving aqueous solutions. The partitioning ratio of MCFAs to SCFAs during the second batch cycle is shown in Figure 4. One can expect MCFA:SCFA partitioning ratios greater than 40:1 several days into a batch cycle. The MCFA:SCFA partitioning ratio will plateau as the extraction process approaches a pseudo-steady state. If ratios &#62;40 cannot be achieved after several days, this suggests the TOPO extractant has degraded or eluted. If this occurs, a new extraction solution should be prepared (see&#160;step 1.1). If the ratio decreases following the plateau phase, this suggests the MCFAs have accumulated beyond 80% of their saturation point. If this occurs, a new stripping solution should be prepared (see step 1.4)
Low MCFA extraction efficiency can be caused by insufficient flow rates within the LLE system. In Figure 5, the pumping speed was reduced in the fermentation broth and stripping solution circulation line to illustrate the impact of diminished liquid recirculation rates on MCFA extraction efficiency. Extraction efficiency is defined as the percentage of MCFA extracted in the stripping solution relative to the total MCFAs produced by the bioreactor plus the MCFAs extracted by the LLE. One can expect extraction efficiencies greater than 85% during normal operation (Figure 5, Day 1-14). When the pump speed is low (Figure 5, Day 14), the extraction efficiency decreases in response. When an adequate pump speed is restored, it can take several days for the extraction efficiency to recover. This could be caused by a reduction in the steady-state concentration of MCFAs in the extraction solution caused by the differential in the extraction rates of the stripping solution (higher) than the fermentation broth (lower).
Several other factors can contribute to diminished extraction efficiencies, including (1) membrane fouling, (2) restricted fluid flow at each stage of the LLE system due to blockages, (3) the formation of gas pockets in the membrane contactors, and (4) allowing the MCFA concentrations in the stripping solution to approach their saturation points. Membrane fouling is indicated by a reduction of membrane flux over time relative to initial conditions. While biofilm formation is unlikely in the FEM, fouling can occur due to the accumulation of cell debris and other suspended solids. Also, while the BEM is aseptic, flow can be obstructed due to the precipitation of fatty acid salts within the membrane contactor or tubing over time. However, routine maintenance and cleaning of the membrane contactors (see step 2.6) should preclude fouling and salt precipitation issues from developing. Gas pockets sometimes form on the top shell side of the membrane contactors due to improper positioning. The membrane contactors should be slightly tilted from vertical to ensure that the shell-side outlet port is at the highest point, allowing any gas that forms to escape the contactor. Fluid flow in the LLE system is configured to flow from the bottom to the top of the contractors to help flush out gas pockets. Finally, MCFA transfer from the extraction solution to the stripping solution in the BEM is diminished at very high MCFA concentrations in the stripping solution. This issue can be remedied by replacing the stripping solution more frequently.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66956/66956fig01.jpg" /> 
Figure 1: Overview of the liquid-liquid extraction system. A diagrammatic rendering showing the major system components, the various fluid circuits, and the flow directions. The major system components are labeled as follows: (A) organic-phase extraction solution reservoir, (B) forward exchange membrane, (C) backward exchange membrane, (D) extraction solution diaphragm pump, (E) aqueous-phase stripping solution reservoir, (F) pH-control system, (G) bioreactor out-flow port, (H) bioreactor return-flow port, (I) forward exchange membrane and hollow-fiber membrane filter peristaltic pump, (J) hollow-fiber membrane filter, (K) pressure gauge, (L) needle valve, and (M) stripping solution peristaltic pump. <a href="https://www.jove.com/files/ftp_upload/66956/66956fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66956/66956fig02.jpg" /> 
Figure 2: Fatty acid accumulation in the stripping solution. Data showing the short-chain fatty acids and medium-chain fatty acid concentrations during three batch cycles of the stripping solution during continuous liquid-liquid extraction operation. <a href="https://www.jove.com/files/ftp_upload/66956/66956fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66956/66956fig03.jpg" /> 
Figure 3: Stripping solution color change after extraction. A photograph showing the color change of the aqueous-phase stripping solution before (i.e., pre-batch) and after (i.e., post-batch)&#160;a batch cycle. <a href="https://www.jove.com/files/ftp_upload/66956/66956fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66956/66956fig04.jpg" /> 
Figure 4: Fatty acid ratios in the stripping solution. Data showing the ratio of medium-chain fatty acids to short-chain fatty acids during a batch cycle of the stripping solution during continuous liquid-liquid extraction operation. <a href="https://www.jove.com/files/ftp_upload/66956/66956fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66956/66956fig05.jpg" /> 
Figure 5: Effect of membrane flow rates on extraction efficiency. Data showing the effect of insufficient flow rates through the forward and backward exchange membrane on medium-chain fatty acid extraction efficiency during operation. <a href="https://www.jove.com/files/ftp_upload/66956/66956fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Optimizing Hollow-Fiber Membranes for Continuous Liquid-Liquid Extraction of Medium-Chain Fatty Acids

A liquid-liquid extraction (LLE) system involving hollow-fiber membranes was developed to continuously and selectively extract medium-chain fatty acids (MCFAs) from the fermentation broth. The LLE system achieves high MCFA specificities from broths containing short-chain fatty acids and alcohols. Also, MCFAs are concentrated in a stripping solution to facilitate product recovery.

Continuous Liquid-Liquid Extraction of Medium-Chain Fatty Acids from Fermentation Broth Using Hollow-Fiber Membranes

Medium-chain fatty acids (MCFAs; carbon-lengths: C6-C12) are high-value platform chemicals that serve a variety of industrial applications, including ...

continuous-liquid-liquid-extraction-medium-chain-fatty-acids-from

Research

JoVE Journal

Bioengineering

966 Views.  University of Georgia. A liquid-liquid extraction (LLE) system involving hollow-fiber membranes was developed to continuously and selectively extract medium-chain fatty acids (MCFAs) from the fermentation broth. The LLE system achieves high MCFA specificities from broths containing short-chain fatty acids and alcohols. Also, MCFAs are concentrated in a stripping solution to facilitate product recovery. 

Video: Author Spotlight: Optimizing Hollow-Fiber Membranes for Continuous Liquid-Liquid Extraction of Medium-Chain Fatty Acids

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 ...

Environment

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

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.

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.

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

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Biochemistry

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Biology

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Solid-Liquid Extraction

Source: Laboratory of Dr. Jay Deiner &#8212; City University of New York
Extraction is a crucial step in most chemical analyses. It entails removing the analyte from its sample matrix and passing it into the phase required for spectroscopic or chromatographic identification and quantification. When the sample is a solid and the required phase for analysis is a liquid, the process is called solid-liquid extraction. A simple and broadly applicable form of solid-liquid extraction entails combining the solid with a solvent in which the analyte is soluble. Through agitation, the analyte partitions into the liquid phase, which may then be separated from the solid through filtration. The choice of solvent must be made based on the solubility of the target analyte, and on the balance of cost, safety, and environmental concerns.

Solid-Liquid Extraction: Principle, Procedure, Applications

Source: Laboratory of Dr. Jay Deiner &#8212; City University of New York
Extraction is a crucial step in most chemical analyses. It entails removing the ...

Continuous Liquid-Liquid Extraction of Medium-Chain Fatty Acids from Fermentation Broth Using Hollow-Fiber Membranes

Summary

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Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

Solid-Liquid Extraction