initiating the operation of the liquid-liquid extraction system for continuous medium chain fatty acid removal

Sustainable MCFA Production via Chain Elongation and Liquid-Liquid Extraction

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.

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

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

My research aimed to evaluate the efficiency of hollow-fiber membrane and the continuous liquid-liquid extraction of medium-chain fatty acid from fermentation broth. I seek to understand how this membrane differs from traditional techniques by optimizing the extraction process and determination of the scalability of this method for industrial use. Current experimental challenge include inefficient extraction of medium-chain fatty acid from fermentation broth, which often lead to the inhibition of the chain-elongation microbes.Traditional techniques often face issues like batch-processing limitation, high energy consumption, and high solvent usage. Our protocol offers several advantages over traditional techniques. These include high selectivity and efficiency in extracting the medium-chain fatty acids from fermentation broths which leads to reduced solvent usage and continuous operation from batch processing.Additionally, this method can easily be replicated for diverse applications.

Initiating the Operation of the Liquid-Liquid Extraction System for Continuous Medium Chain Fatty Acid Removal

To begin, integrate the bioreactor and liquid-liquid extraction system. Turn on the stripping solution peristaltic pump, or backward extraction membrane, and set the pump speed to achieve a constant flow rate between 25 to 250 milliliters per minute. Slowly close the needle valve at the shell-side outlet of the backward extraction membrane to establish a back pressure.Then, turn on the first hydrophobic hollow-fiber membrane module peristaltic pump, and set the pump speed to achieve a constant flow rate between 25 to 250 milliliters per minute. Afterward, slowly close the needle valve at the shell-side outlet of the first hydrophobic hollow-fiber membrane module to establish a back pressure of approximately five pounds per square inch gauge. Now, turn on the organic phase extraction solution diaphragm pump, and set the pump speed to achieve a constant flow rate between five to 50 milliliters per minute.Lastly, check the system for continuous flow. A steady accumulation of short and medium-chain fatty-acid products was observed in the alkaline aqueous phase stripping solution. A reduction in pump speed lowered the extraction efficiency.After restoring adequate pump speed, efficiency may take several days to recover.

initiating-operation-liquid-liquid-extraction-system-for-continuous

Research

JoVE Journal

Bioengineering

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.

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.

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

Constructing and Integrating the Bioreactor and the Liquid-Liquid Extraction System

To begin, prepare the organic phase extraction solution and reservoir by adding the extraction solution to a two liter glass reservoir and placing it on a magnetic stir plate. Next, position a 100 RPM variable speed pump drive with a polytetrafluoroethylene diaphragm pump head next to the reservoir. Using flexible pump tubing, connect the outflow port of the extraction solution reservoir to the diaphragm pump inlet.Then connect the pump outlet to the shell-side inlet at the base of the forward extraction membrane. Now, connect the shell-side outlet at the top of the forward extraction membrane to the shell-side inlet located at the base of the backward extraction membrane. Then connect the shell-side outlet at the top of the backward extraction membrane to the return port on the extraction solution reservoir.To prepare the aqueous phase stripping solution and reservoir, pour the solution into a four liter glass reservoir with a magnetic stir bar and place it on a magnetic stir plate. Integrate a pH control system with a set point of pH nine with the stripping solution reservoir. Insert the pH probe through the stripping solution reservoir port and suspend it in the stripping solution.To install the hollow fiber membrane filter, place a 300 RPM variable speed pump drive adjacent to the bioreactor. Affix the hydrophilic hollow fiber membrane to a ring stand above the peristaltic pump. Stack two peristaltic pump heads, large and small, on the pump drive.Now using flexible tubing, connect the outflow port at the bioreactor to the large pump head inlet. Then connect the large pump head outlet to the hydrophilic hollow fiber membrane filter at the tube-side inlet located at the base of the filter. Cap the top shell-side port of the filter to prevent the influx of air.To connect the forward extraction membrane, affix the first hydrophobic hollow fiber membrane module to a ring stand above the pump. Using flexible pump tubing, connect the hydrophilic hollow fiber membrane filter at the shell-side outlet to the small pump head inlet. Then connect the small pump head outlet to the forward extraction membrane at the tube-side inlet located at the base of the module.Now using a coupling fitting and T fitting, connect the pressure gauge and flow restriction valve to the tube-side outlet of the forward extraction membrane. Next, connect the tube-side outlet of the forward extraction membrane to the return port at the bioreactor. To connect the backward extraction membrane, place a 300 RPM variable speed pump drive adjacent to the bioreactor.Affix the second hydrophobic hollow fiber membrane module to a ring stand above the peristaltic pump. Then using flexible pump tubing, connect the stripping solution reservoir outflow port to the peristaltic pump inlet and the pump outlet to the tube-side inlet at the base of the backward extraction membrane. Afterward, using a coupling fitting and T fitting, connect the pressure gauge and flow restriction valve to the tube-side outlet of the backward extraction membrane.Using flexible pump tubing, connect the tube-side outlet at the top of the backward extraction membrane to the stripping solution reservoir return flow port.