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 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. 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.
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 β-oxidation reaction that would normally break down longer chain carboxylates to acetate (e.g., C6 → C4 → C2) can be reversed (e.g., C2 → C4 → 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 <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.
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 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., 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' 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.
The authors have nothing to disclose.
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.
10 L Media Bottle | Duran | 218018658 | |
3.5 L Media Bottle | Duran | 218016957 | |
Boric acid, 99.5%, | ThermoScientific (Fisher Scientific) | 327132500 | |
Hydrophilic MINIKROS 20CM 0.2UM PES 1MM 1.5TC X 3/4TC | Repligen | N02-P20U-10-N | |
L/S Variable-Speed Pump Drive; 100 rpm | MasterFlex (VWR) | MFLX07528-10 | |
L/S Variable-Speed Pump Drive; 300 rpm | MasterFlex (VWR) | MFLX07528-20 | |
Light Mineral Oil, NF (4 Liters) (CAS: 8042-47-5) | Thomas Scientific | C761Z18 | |
Liqui-Cel 2.5×8 X50 membrane CO2, PP Housing Viton O-rings (0.5-3 gpm (0.1-0.7 m3/h)), 1/4-in FNPT connections | 3M | LC-02508X50-G453 | |
Magnetic Stirrer, 20 L Capacity, 110 V | Cole-Parmer | EW-04661-29 | |
Masterflex L/S Precision Pump Tubing, Tygon, Size 14 | MasterFlex (VWR) | MFLX06402-14 | Specific tubing size will depend on application. |
Masterflex L/S Precision Pump Tubing, Tygon, Size 16 | MasterFlex (VWR) | MFLX06402-16 | Specific tubing size will depend on application. |
Masterflex L/S Precision Pump Tubing, Tygon, Size 17 | MasterFlex (VWR) | MFLX06402-17 | Specific tubing size will depend on application. |
Masterflex L/S Precision Pump Tubing, Tygon, Size 18 | MasterFlex (VWR) | MFLX06402-18 | Specific tubing size will depend on application. |
MasterFlex L/S Standard Pump Head for Precision Tubing L/S 14, Polycarbonate Housing, CRS Rotor | MasterFlex (VWR) | MFLX07014-20 | Specific pump head size will depend on application. |
MasterFlex L/S Standard Pump Head for Precision Tubing L/S 14, Polycarbonate Housing, CRS Rotor | MasterFlex (VWR) | MFLX07014-20 | Specific pump head size will depend on application. |
MasterFlex L/S Standard Pump Head for Precision Tubing L/S 16, Polycarbonate Housing, CRS Rotor | MasterFlex (VWR) | MFLX07016-20 | Specific pump head size will depend on application. |
MasterFlex L/S Standard Pump Head for Precision Tubing L/S 17, Polycarbonate Housing, CRS Rotor | MasterFlex (VWR) | MFLX07017-20 | Specific pump head size will depend on application. |
MasterFlex L/S Standard Pump Head for Precision Tubing L/S 18, Polycarbonate Housing, CRS Rotor | MasterFlex (VWR) | MFLX07018-20 | Specific pump head size will depend on application. |
MasterFlex PTFE-diaphragm pump head, 10 to 100 mL/min | MasterFlex (VWR) | MFLX07090-62 | |
Oakton 220 pH/ORP/Temperature Controller, 1/8 DIN | Spectrum Laboratory Products | 664-12595-E1 | |
Oakton 220 pH/ORP/Temperature Controller, 1/8 DIN | Spectrum Laboratory Products | 664-12595-E1 | |
Oakton Female BNC-to-Stripped Wire Adapter | Spectrum Laboratory Products | 664-12592-E1 | |
pH Probe with BNC Connector | ThermoScientific | 10010-788 | Any pH probe with a BNC connector will suffice. |
Precision Flow-Adjustment Valve, White Polypropylene, 1/4 NPT Male x Male | McMaster-Carr | 7792K57 | |
ProConnex Fittings Kits – A | Repligen | ACPX-KT2-01N | Compatible with Hydrophilic MINIKROS Filter |
ProConnex Fittings Kits – B | Repligen | ACPX-KT1-01N | Compatible with Hydrophilic MINIKROS Filter |
Sodium Hydroxide Pellets for Analysis | Sigma Aldrich | 1.06498 | |
Stainless-Steel Pressure Gauge 0-60 psi Stainless Steel 1/4" NPT 2.5" Face Dial | NA | XJ-219 | Any comparable pressure gauge covering 0-60 psig range will suffice. |
Trioctylphosphine oxide (TOPO) | Sigma-Aldrich | 346187-100G |
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