Green Solvent-Based Approaches for Volatile Fatty Acid Production and Recovery from Organic Waste

Abstract

Volatile fatty acids (VFAs) are essential precursors in chemical synthesis for various chemicals, polymers, pharmaceuticals, and fragrance compounds. Acidogenic anaerobic digestion (or arrested methanogenesis) is a promising method to stabilize organic wastes and convert them to value-added products such as VFAs. However, the VFAs’ accumulation could in turn suppress the fermentation process through product inhibition and limit the titer of VFA in the digestate. Therefore, in situ separation and recovery of VFAs from the fermentate is crucial to constructing an effective continuous VFA-producing system. Recent research has been dedicated to addressing these issues and advancing the utilization of biobased VFAs, particularly through process-intensified strategies employing novel green solvents such as natural deep eutectic solvents. Furthermore, in situ conversion of VFAs into esters is another potential strategy for VFA removal. However, VFA esterification in an aqueous medium is challenging due to the abundant water driving the reaction toward hydrolysis. Recent advances in free or immobilized enzyme catalysis in solvents have demonstrated improved ester yield by providing a hydrophobic space for the esterification reaction in aqueous solution. In this review, we present an overview of critical aspects on the state-of-the-art of green solvent-based process intensification strategies, including feedstock selection and pretreatment, operating condition optimization, advances in membrane- and solvent-based recovery methods, and biocatalytic in situ esterification. Lastly, we provide perspectives toward cost-effective, continuous, high-solid, environmental-benign, and industrial-relevant VFA production applications.

1. Introduction

Volatile fatty acids (VFAs), typically defined as short-chain fatty acids with carbon chain lengths ranging from C2 to C6, are key platform chemicals with wide applications in the production of chemicals, polymers, pharmaceuticals, and fragrance compounds [1]. The sustainable production of VFAs from organic waste through anaerobic fermentation has attracted increasing attention as an environmentally friendly and economically viable alternative to fossil-based processes. In particular, the conversion of heterogeneous biowaste streams, such as organic solid waste, wastewater sludge, and lignocellulosic residues, into VFAs offers a promising pathway for waste valorization and circular bioeconomy development. Despite these advantages, several challenges hinder the efficient production and utilization of VFAs. One of the primary limitations is the relatively low concentration of VFAs (typically 1–2%) in fermentation broths, which significantly increases the recovery cost and complexity of downstream separation and purification [2,3]. In addition, the accumulation of VFAs during fermentation can lead to product inhibition, lowering microbial activity and limiting overall productivity. These challenges highlight the need for integrated strategies that simultaneously enhance VFA production, mitigate inhibition effects, and enable efficient recovery and upgrading.

Extensive efforts have been devoted to improving individual stages of the VFA production process, including feedstock pretreatment, optimization of fermentation conditions, and development of recovery technologies. However, most existing studies focus on single-process steps, with limited integration between upstream production and downstream separation. In particular, strategies that couple VFA production with in situ removal and subsequent valorization remain underexplored. Moreover, while conventional recovery methods such as distillation, adsorption, and membrane separation have been widely investigated, they often suffer from high energy consumption, limited selectivity, or operational complexity.

In recent years, process intensification (PI) has emerged as a promising approach to address these limitations by integrating multiple process functions, improving mass and heat transfer, and enhancing overall system efficiency [4]. Within this context, green solvents, especially deep eutectic solvents (DESs), have gained increasing attentions due to their tunable physicochemical properties, low volatility, and potential biocompatibility. DES is a liquid eutectic mixture comprising two or more constituents, commonly a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) [5]. The mixture exhibits a melting point (usually below room temperature) lower than that of its individual components. The formation of a DES and the reduction in melting temperature are generally attributed to the inter- and intramolecular interactions among the constituents, including hydrogen bonding, van der Waals interactions, and ionic bonding [6].

There has been a growing exploration of specific DESs, particularly natural product-based DES (NADES), as promising green solvents [7]. This is attributed to their desirable properties, such as good biomass solubilizing ability, extractability, biocompatibility, and low toxicity [6]. The physicochemical properties of a DES are determined by its components and their synergetic effect. Recent advancements have been made in the development of hydrophobic DESs through careful compound selection. The hydrophobic nature of these DESs made them as excellent extractants for VFAs and a wide range of bioactive compounds including artemisinin, polyprenyl acetates, baicalin and phytocannabinoids, among others [8]. These characteristics make DESs suitable for integration with acidic fermentation at different stages, such as feedstock pretreatment, VFA extraction, and downstream esterification processes. Additionally, these solvents offer new opportunities for integrating VFA production, extraction, and upgrading into a single system. In particular, hydrophobic DESs have demonstrated potential for selective VFA extraction, while enzyme-compatible DES systems enable in situ esterification as a downstream valorization strategy.

In this review, we present a comprehensive overview of process intensification strategies for enhancing VFA production from organic wastes, with particular emphasis on the integration of production, recovery, and valorization pathways. We begin by examining approaches to improve VFA generation through acidic fermentation, including methanogenesis inhibition, feedstock pretreatment, and optimization of key operational parameters. We then summarize recent advances in VFA recovery technologies that enable efficient separation and mitigate product inhibition. Particular attention is given to the emerging role of DESs as multifunctional platforms, serving as pretreatment agents, extractants, and reaction media for downstream esterification. Finally, we discuss the key challenges and future research directions toward the development of cost-effective, continuous, and sustainable VFA production systems, highlighting the importance of integrated process design in advancing VFA-based biorefineries.

2. Basics of Arrested Methanogenesis

Arresting methanogenesis (AM), commonly referred to as arrested anaerobic digestion (AAD), is a key strategy for enhancing VFA production by preventing the conversion of the intermediates into biogas. In conventional AD, VFAs are produced during hydrolysis and acidogenesis and subsequently consumed during methanogenesis via hydrogenotrophic (H₂/CO₂) and acetoclastic (acetate) pathways. As such, VFAs serve as central intermediates that are ultimately converted into methane. In an AAD process, methanogenic activity is suppressed or eliminated, which halts VFA consumption and enables their accumulation. The elevated VFA concentrations further inhibit methanogenesis through pH reduction and the toxicity of undissociated acids, creating a self-reinforcing acidic fermentation environment. Various strategies have been explored and reviewed [9,10,11] to achieve AAD, including (i) the addition of methanogen-specific inhibitors such as 2-bromoethanesulfonate and antibiotics, (ii) inoculum pretreatment via acid shock or thermal treatment to deactivate methanogens, and (iii) process control strategies that favor acidogenesis, such as high organic loading rates, short hydraulic retention times, and low-pH conditions [9]. For example, in situ generation of H₂O₂ has been shown to selectively inhibit methanogens, resulting in VFA accumulation up to 10.6 g COD L⁻¹ (and 26.7 g COD L⁻¹ after recovery) while preserving acidogenic activity [12]. Similarly, an integrated upstream process combining alkaline pretreatment with AAD achieved a VFA titer of 3.6 g COD L⁻¹ at pH 10, which was approximately 23% lower than the VFA titer reported for systems employing chemical methanogenesis inhibitors [13].

To further enhance VFA production from complex organic wastes, feedstock pretreatment is essential to disrupt recalcitrant structures, improve solubilization, and increase substrate bioavailability for hydrolytic and acidogenic microorganisms. However, the accumulation of VFAs can impose product inhibition, limiting microbial activity and overall conversion efficiency. Integrating in situ VFA removal strategies such as membrane extraction, adsorption, or liquid–liquid extraction can alleviate this inhibition by maintaining favorable fermentation conditions. Moreover, coupling in situ esterification enables simultaneous conversion of VFAs into value-added esters, reducing acid toxicity while improving product recovery and process economics. The performance of AAD systems is also strongly influenced by microbial community structure, hydrogen partial pressure, and fermentation-associated environmental stresses. Previous studies have shown that acidogenic consortia are typically dominated by fermentative bacterial groups such as Clostridium, Firmicutes, and Bacteroidetes [14], while suppression of methanogenic archaea is critical for sustained VFA accumulation [15]. In addition, environmental factors including pH, oxidation–reduction potential, and undissociated VFA concentration can significantly affect metabolic pathways and product selectivity [16]. Detailed discussions regarding microbial ecology [17], syntrophic interactions, inhibition thresholds, and omics-based analyses [18] of AAD systems have been comprehensively reviewed elsewhere and are therefore not extensively discussed in this review. To manipulate these biological and physicochemical constraints, a variety of AAD process intensification strategies have been developed, ranging from conventional operational controls to more advanced approaches that directly influence microbial metabolism and product recovery. Traditional AAD strategies, including pH regulation, inoculum pretreatment, chemical inhibition, and operational control through organic loading rate or hydraulic retention time [19,20], are generally attractive because of their simplicity, low implementation cost, and compatibility with existing anaerobic digestion infrastructure. However, these approaches often provide limited control over VFA selectivity and may suffer from process instability under variable feedstock conditions. In contrast, advanced strategies such as bioaugmentation [21], electro-fermentation [22], and in situ product recovery [23] offer greater control over microbial metabolism, redox balance, and product accumulation, resulting in improved VFA productivity and selectivity. These approaches are particularly advantageous when high-value VFA streams, continuous operation, or downstream upgrading are desired, although they typically require higher capital investment and operational complexity. More importantly, integrating VFA production with in situ recovery and upgrading technologies can mitigate product inhibition, stabilize microbial activity, and improve overall carbon utilization efficiency, thereby addressing limitations that cannot be fully resolved by fermentation control alone. Together, these approaches provide a synergistic pathway to intensify AAD systems, enhancing carbon flux toward VFAs and enabling more stable and efficient waste valorization processes. The following sections critically reviewed each topic area which led to perspectives for further development of an integrated VFA production system.

3. Feedstock Pretreatment

Various types of biowaste have been studied as feedstock for AAD, including wastewaters, wastewater sludge, the organic fraction of municipal solid waste, residues from biodiesel production, dairy industrial waste, and lignocellulosic biomass from different sources [24]. The composition of the feedstock has a significant impact on the production and distribution of fermentation products. For instance, a high proportion of carbohydrates can enhance acetate generation, while the protein content in the substrate has been found to influence propionate production [25]. Lignocellulosic feedstock, including plant-based dry matter or biomass such as forest residues, distillery spent grains, yard waste, and various agricultural wastes like wheat straw, bagasse, among others, has been extensively studied due to its abundant availability and relatively cost-effective nature [24]. Lignocellulosic feedstock typically requires a pretreatment step aiming to improve the accessibility and reactivity of the polysaccharides by disrupting their intricate three-dimensional structure and removing the lignin fraction that inhibits enzymatic hydrolysis, while minimizing polysaccharide degradation [26]. Various pretreatment methods have been developed [9], including mechanical (e.g., milling, grinding, and chipping), chemical (e.g., acid, alkali, solvent, and ozonation), physical (e.g., thermal treatment, microwave irradiation, and ultrasound treatment) and biological treatments involving the addition of microorganisms or enzymes [10].

Among the solvent-based methods, DES-based pretreatment has attracted increasing attention in recent years. Certain DESs, such as those composed of choline chloride or lactic acid, have been observed to lower the cellulose crystallinity, thereby increasing the accessible reaction area [26]. Additionally, pretreatment with DESs containing choline chloride has also been found to facilitate the cleavage of β-O-4 bonds in lignin, resulting in depolymerization and dissolution of lignin [27,28].

The effectiveness of DES pretreatment relies on its composition and pH. Acidic DESs, composed typically of organic carboxylic acids acting as HBD, and basic DESs, containing amine or amide groups, are the most preferred types of DES for biomass pretreatment. Neutral DESs, on the other hand, are generally not efficient for biomass fractionation. Nevertheless, certain hydrophilic Type III DESs, particularly polyol-based systems such as choline chloride/glycerol and choline chloride/ethylene glycol, exhibit excellent compatibility with enzymes and microorganisms, making them promising options for simultaneous saccharification and biological pretreatment [6]. For instance, DESs containing glycerol (Gly) or ethylene glycol (EG) as HBD and combined with choline chloride (ChCl), a less toxic HBA candidate, have exhibited favorable biocompatibility with cellulase [29,30]. After incubation in 10% (v/v) ChCl/Gly (1:2) or ChCl/EG (1:2) solutions for 24 h, the enzyme retained over 90% of its initial activity [31]. In fact, numerous DESs, irrespective of their pH, have exhibited favorable compatibility with hydrolytic enzymes and have been extensively studied in combination with enzymatic hydrolysis (EH) pretreatment [31,32,33]. Ionic liquids (ILs), considered as a predecessor or subcategory of DESs, often exhibit inhibitory effects on commercially hydrolytic enzymes, necessitating the excessive use of water for detoxification after biomass pretreatment [34]. Conversely, choline-based NADES show good compatibility with enzymes. As summarized in

Table 1. DES pretreatment followed by enzymatic hydrolysis (modified from Ullah et al., 2022 [6]).

Substrate	DES Type and Pretreatment Condition	Enzyme Type and EH Condition		DES in EH	EH Result *	Reference
Washing-eliminated strategy
Sugarcane bagasse	ChCl/malonic acid (1:1). 4% solid loading, 130 °C, 3.2 h	Cellulysin® Cellulase Trichoderma viride 30 FPU/g; 2 wt% solid loading, pH 4.8, 50 °C, 48 h.		<1%	7.1 g/L glucose	[32]
				~0	7.2 g/L glucose
bamboo residue	ChCl/ethanolamine (1:6). 6.25% (w/w) solid loading, microwave 130 °C, 10 min	Cellulase (Cellic® CTec2, 100 FPU/mL). 15 FPU/g enzyme loadings, 72 h.		NA	GY 88.3%	[35]
oil palm empty fruit bunch	ChCl/Lactic acid (LA) (1:2); ChCl/Gly (1:2); ChCl/Urea (U) (1:2). Solid-to-liquid ratio 1:10 (w/v), 120 °C, 3 h	Cellulase from Trichoderma viride, 10 mg/mL in buffer. Solid-to-liquid ratio of 2:1, 50 mM, pH4.8, 50 °C, 48 h.		NA	RSY for ChCl/LA (1:2), ChCl/Gly (1:2), ChCl/U (1:2): 20.7%, 20.0%, 16.9%	[36]
Rice straw	ChCl/lactic acid (1:5). 5% solids loading, 60 °C, 12 h	Crude cellulase from Aspergillus terreus D34, 9 FPU/g. 10% solids loading, 42 °C, 24 h.		NA	RSY 333 mg/g	[37]
Wheat straw	ChCl/acetic acid (1:2); optimized conditions of 139.6 °C for 3 h 47 min; 1:10 solid-to-liquid ratio	Cellic® CTec2; 15% (w/v) biomass loading; 15.45 FPU/g glucan; 50 mM citrate buffer, pH 4.8; 50 °C, 125 rpm, 72 h.		NA	Washed and unwashed biomass showed similar hydrolysis: glucose 10.18 vs. 9.85 g/L and glucan-to-glucose yield 27.13 vs. 26.08 wt%; xylose 2.38 vs. 2.91 g/L and xylan-to-xylose yield 25.73 vs. 29.96 wt% at 72 h	[38]
One-pot process
corn stover	ChCl/glycerol (1:2). 10% (w/v) solid loading, 180 °C, 2 h~8 h	Cellic® CTec2 and HTec2. Dilute with citrate buffer, 0.05 M, pH 5, 20 mg/g glucan; 2 mg/g xylan; 50 °C, 3 d.		11.25% (w/v)	GY 84.5%	[30]
Xylan, dissolve in DES	ChCl/U (2:1, 1:1, 1:2)	Cellic® Tec2, 4800 UI/g xylan. pH 4.8, 50 °C.	20 g/L xylan	7%	XY 60~82%	[39]
			50 g/L xylan	17%	XY 50~76%
			100 g/L xylan	33%	XY 35~55%
Rice husk	ChCl/Gly (1:2); ChCl/EG (1:2). 4% (w/v) solid loading 115 °C, 3 h	Cellulases from Aspergillus sp., 1000 units/g. Dilute with 0.05 M citrate buffer, pH 4.8, 50 °C, 3 h.		10% (v/v)	GY 0.7 mM, enhance > 180% than unpretreated sample	[31]
Rice husk	ChCl/Gly (2:1); ChCl/EG (2:1). 10% (w/v) solid loading, 115 °C, 12 h	Halophilic cellulase from Aspergillus terreus UniMAP AA-6, 0.4 U/mL. Dilute with 0.05 M citrate buffer, pH 4.8, 50 °C, 36 h.		20% (v/v)	more than 1 mM glucose yield; 2~3 folds increase than untreated sample	[40]
Parthenium hysterophorus biomass	ChCl/Sorbitol (1:5, Ultrasonic radiation combined) 5 wt% solid concentration in DES, 50% amplitude, 20 min, 40 W, 20 kHZ; ChCl/sorbitol (1:2 and 1:5); ChCl/oxalic acid (1:2); ChCl/Gly (1:2); ChCl/U (1:2); ChCl/imidazole (1:2); ChCl/succinic acid (1:2); ChCl/LA (1:2). 5 wt% solid concentration, 121 °C, 15 min	Enzyme from A. aculeatus PN14, FPU 40 IU/g; xylanase 861 IU/g biomass. Directly in the same reaction tube; 50 °C, 72 h.		100%	RSY (mg/g): ChCl/sorbitol (1:5, ultrasonic combined) = 233; ChCl/sorbitol (1:2) = 114; ChCl/sorbitol (1:5) = 149; ChCl/oxalic acid (1:2) = 101; ChCl/Gly (1:2) = 83; ChCl/U (1:2) = 76; ChCl/imidazole (1:2) = 67; ChCl/succinic acid (1:2) = 62; ChCl/LA (1:2) = 58	[33]
Rice straw	ChCl/LA (1:3), containing 10% (w/w) water, 15% biomass loading, 120 °C for 3 h.	20 FPU Celluclast 1.5 L/g solid, 0.1% Viscozyme L.	1.5% (w/w) dry biomass sample, citrate buffer, pH 4.8	~0	TSY 49.9%	[41]
		40 FPU enzymes/g solid fractions.		0.3 mol/L	TSY 75.7%
Rubber seed shell	ChCl/formic acid (ChCl:FA, 1:5; 120 °C, 2 h) or potassium carbonate/glycerol (PC:Gly, 1:9; 130 °C, 6 h); 5 wt% solid loading	Commercial cellulase (30 FPU/g) and xylanase (20 U/g); citrate buffer (50 mM, pH 4.8 for cellulase and pH 5.3 for xylanase; 50 °C, 150 rpm, 48 h.		For cellulase, 2% (v/v) ChCl:FA or PC:Gly; for xylanase, 4% (v/v) ChCl:FA	PC:Gly gave higher TSY (0.38 g/g) than ChCl:FA (0.01 g/g)	[42]
Napier grass	ChCl/sorbitol (1:2), ChCl/urea (1:2), or ChCl/lactic acid (1:4); pretreatment conditions optimized across 90–130 °C, 1–3 h, and 5–15 wt% biomass loading	Cellic® CTec2 cellulase; 30 FPU/g biomass; 50 °C, 72 h.		5% (v/v) DES	Glucose yield: 379.00 mg/g (ChCl:Sorbitol), 396.01 mg/g (ChCl:Urea), and 385.42 mg/g (ChCl:LA)	[43]

* EH: enzymatic hydrolysis; GY: glucose yield; XY: xylose yield (g xylose/100 g xylan); RSY: reducing sugar yield; TSY: total sugar yield. Values are reported in their original forms from the cited studies because different studies used different hydrolysis performance metrics and calculation methods.

, NADES allow for the elimination of the washing steps or even enable a one-pot delignification and EH/saccharification process, thereby avoiding excessive water usage and related waste disposal issues [6].

4. VFA Extraction

The production of VFAs through AAD is constrained by two major challenges. First, VFAs are typically present at relatively low concentrations in the fermentation broth, which limits their practical utilization and poses significant challenges for downstream recovery. Second, the accumulation of VFAs can lead to product inhibition, as the decrease in pH and the increased concentration of undissociated acids adversely affect microbial activity and metabolic stability (Figure 1). These challenges highlight the need for strategies that increase VFA concentration, suppress methanogenesis, and alleviate product inhibition to achieve efficient and stable fermentation processes. As a result, in situ removal/extraction of VFAs from fermentation broth is an essential step in ensuring the feasibility of the entire VFA production chain.

Table 2. Overview of common VFA recovery methods.

Method	Mechanism	Typical Conditions	Pre-/Post-Treatment	Development Stage	Main Advantages	Main Limitations	Ref.
Adsorption	VFAs bind to adsorbents via ion exchange or hydrophobic interactions.	Adsorbents such as amine-based anion exchange resins.	Broth clarification; desorption with solvents (ethanol, NaOH, etc.) or heat post-treatment.	Pilot-to-commercial stage.	Cost-effective; flexible across pH conditions; scalable.	Regeneration complexity; sensitivity to competing ions; resin selection is critical.	[44,45,46,47,48]
Distillation	Separation based on differences in boiling points.	Dewatering the fermentation broth, followed by further distillation to recover the VFAs in the distillate; reactive distillation may enhance volatility and separability.	Requires pre-concentration; acidification usually needed.	Commercial stage, commonly used for solvent recovery and VFA concentration in downstream processing.	Well-established industrial technology; capable of achieving high product purity.	Extremely energy-intensive; inefficient for dilute VFA streams.	[10,49]
Electrodialysis	VFAs migrate through ion-exchange membranes under an electric field.	Ion-exchange membranes operated between electrodes; most effective when VFAs are dissociated.	Solids removal to prevent fouling; often needs pH staging, acidification, or downstream acid/salt conversion.	Applied research and pilot-scale development stage.	Operating at fermentation pH is allowed; high recovery and selectivity; reduced chemical consumption; compatible with in situ recovery.	Membrane fouling; ion competition; high membrane cost.	[50,51,52,53]
Esterification	VFAs are converted to esters	Typically requires alcohols with acid catalysts or enzymes.	Requires pre-concentration of VFAs; downstream ester purification needed.	Late research and pilot-scale development stage.	Produces valuable esters; easier separation of esters than acids; simplified process if ester is the target.	Water sensitivity; low efficiency under dilute conditions.	[54,55,56]
Filtration	Separation based on size and/or charge exclusion using membranes.	Microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO), often used in combination.	MF/UF often used as pre-treatment before other recovery methods; further purification such as extraction, distillation, or adsorption is usually required afterward.	Pilot-to-commercial scaling stage.	High throughput; scalable, low chemical and energy demand.	Membrane fouling; relatively high membrane cost; insufficient as a standalone purification method.	[57,58,59,60,61]
Gas stripping	VFAs are transferred from liquid to gas phase via gas bubbling.	Gas sparging via the fermentation broth; efficiency increases when VFAs are in undissociated form.	Usually requires acidification (pH control) to increase volatility; stripped acids are recovered via condensation or absorption (e.g., alkaline traps).	Advanced pilot stage, with ongoing development for continuous recovery.	Simple operation; less energy consumption; suitable for in situ removal.	Low selectivity; co-stripping of water and other volatiles; inefficient for long-chain VFAs; require large gas volumes; additional recovery steps needed.	[62,63,64]
Liquid–liquid extraction	VFAs partition into a liquid phase that is immiscible with water, driven by chemical potential.	Contact with a water-immiscible organic phase including organic solvents, hydrophobic ILs, and HDES; efficiency increases when VFAs are undissociated; can be enhanced by reactive extractants (e.g., amines like TOA, TOPO).	Solvent regeneration after VFA extraction.	Pilot-commercial stage, with increasing focus on process intensification and industrial implementation.	High efficiency and selectivity; flexible solvent design; capable of producing concentrated organic-phase product.	Solvent toxicity concerns; solvent loss and regeneration challenges; scale-up challenges for IL/DES systems.	[65,66,67,68,69,70]
Membrane contactor	A membrane separates phases, allowing VFAs to diffuse into an extractant or stripping phase.	Uses hydrophobic or omniphobic membranes; generally requires pH < pKa.	Requires broth clarification to prevent fouling; usually acidification (pH < pKa); downstream solvent regeneration needed.	Lab-pilot stage, with growing application in integrated recovery and biorefinery systems.	High selectivity; minimal phase mixing; suitable for in situ removal.	Membrane wetting and fouling; limited membrane lifespan.	[71,72,73,74,75,76]
Pervaporation/membrane distillation	Volatility-driven transport across a membrane under vacuum or temperature gradient.	Functional membranes operated under vacuum or temperature gradient.	Requires pretreatment to prevent fouling; usually employs acidification to enhance volatility; downstream condensation and polishing needed for final product quality.	Lab-pilot stage, with growing interest in energy-efficient separation and concentration.	Strong concentration capability; can utilize low-grade heat.	Relatively high energy consumption; membrane fouling and wetting; challenges in vapor-phase product recovery.	[77,78,79,80]
Salting-out/precipitation	Salt-induced phase separation or precipitation of VFAs.	Inorganic salts (e.g., ammonium sulfate, phosphate salts); often paired with alcohol extractants or alkaline-earth precipitation	Requires salt addition; often followed by solid–liquid separation, phase separation, or filtration; downstream desalting and polishing needed.	Lab-pilot stage, with ongoing development to improve scalability and salt recovery.	Simple operation; no membranes required; high recovery and selectivity achievable.	High salt usage; not suitable for in situ fermentation (may be toxic to microbes); requires salt recovery/recycling; potential corrosion; downstream desalting increases complexity and cost.	[81,82,83]

provides a summary of the different strategies that have been investigated in this field.

From a process intensification perspective, the suitability of each VFA recovery strategy depends not only on extraction efficiency, but also on energy demand, compatibility with continuous fermentation, scalability, solvent recyclability, and operational stability. Conventional approaches such as distillation are industrially mature and capable of producing high-purity products. However, they remain highly energy-intensive for dilute VFA streams. Membrane-based systems and electrodialysis offer advantages for continuous processing and reduced chemical consumption, although membrane fouling and capital costs remain major limitations. In contrast, liquid–liquid extraction using DESs or HDESs has emerged as a promising low-energy alternative due to its tunable selectivity, mild operating conditions, and potential integration with in situ recovery systems. Nevertheless, most DES-based extraction systems are currently at a laboratory scale, and further studies are required to evaluate long-term solvent stability, recyclability, and techno-economic feasibility under industrially relevant conditions.

These strategies employ distinct approaches and possess individual limitations and benefits. Therefore, when selecting a suitable VFA recovery method, several factors must be considered, including effluent properties such as pH value, VFA profile, and composition of other fermentation products, as well as the intended downstream applications. For instance, evaporation-based techniques such as gas stripping and pervaporation are more effective when VFAs remain undissociated. As a result, they are better suited for fermentation effluents with a pH below 4.8 or require an acidification process prior to recovery. On the other hand, methods such as electrodialysis and ion exchange adsorption prefer a neutral or higher pH condition, wherein the enrichment of charged VFA ions is driven by electrostatic effects or an applied potential difference [10]. Upon VFA removal from the fermentation broth, downstream processing becomes necessary to purify and concentrate the VFAs for potential sale in the market or for further upgrading [9]. VFA recovery methods exhibit varying preferences for VFAs with different carbon chain lengths. Hence, the VFA profile and intended downstream application must also be considered when determining the appropriate method to employ [10].

Additionally, it is worth noting that a clarification process is typically required before implementing the discussed VFA recovery methods. Clarification aims to remove solid particles and contaminants from the complex fermentation broth, thereby alleviating their impact on the subsequent VFA recovery steps. Common techniques employed for clarification include centrifugation and filtration, while more advanced separation/condensation methods like electrocoagulation and freeze/thaw separation have also been studied. The separated solid residue is often recycled back to the reactor for further processing to maximize VFA yield and reduce waste generation [10]. Indeed, it has been widely recognized that a preferable VFA recovery process should continuously remove VFAs without disrupting the microbial community, so as to prevent product inhibition and minimize acid-induced stress on the microbes, thereby maintaining a stable microbial performance and enhanced productivity [9].

Among the available recovery strategies, liquid–liquid extraction has emerged as a particularly attractive approach due to its high processing capacity, low energy requirements, operational simplicity, thermal robustness, and the ability to tailor acid selectivity through solvent design [84]. However, the inherently hydrophilic nature of VFAs limits their partitioning into conventional organic solvents such as alcohols, ketones, and esters, resulting in relatively low extraction efficiencies. Consequently, conventional VFA recovery relies on the judicious selection of extractants (e.g., organic amines) and appropriate diluents, along with careful control of key process parameters such as pH and temperature [83]. For instance, tributyl phosphate (TBP) and trioctylamine (TOA), combined with diluents including rapeseed oil, lamp oil, and oleyl alcohol, have been applied for VFA extraction from AD systems using apple pomace and cow dung, achieving recoveries of 2.40 ± 0.30 g L⁻¹ and 5.84 ± 0.36 g L⁻¹, respectively, after 28 days of operation [85]. Similarly, an extraction efficiency of up to 75% was obtained using tri-n-octylphosphine oxide (TOPO, 20%) diluted in kerosene (80%) under optimized conditions [86].

ILs have gained attention as greener alternatives to conventional extractants. For example, Xing et al. [67] developed a [P666,14][Cl]–dodecane system capable of extracting VFAs at near-neutral pH (~6), achieving extraction efficiencies of approximately 50% for acetic acid and over 90% for butyric acid, with enhanced selectivity toward longer-chain VFAs. Likewise, Singh et al. [54] reported a hydrophobic IL system ([P666,14][DBP]) exhibiting high extraction capacities (up to ~843 mg g⁻¹ IL) and a strong preference for longer-chain acids. Despite these advances, ILs still face challenges related to biocompatibility with microbes and enzymes.

More recently, DESs have emerged as a promising alternative, offering simple and cheap preparation, tunable properties, and improved environmental compatibility for VFA extraction. The DES-based process offers a promising approach to address several key challenges in VFA fermentation. First, because VFAs are typically present at low concentrations in fermentation broth, selective extraction into a DES phase can enrich the target products and improve their recoverability. Second, continuous or in situ extraction helps prevent the further consumption of VFAs by reducing their residence time in the aqueous phase, thereby helping preserve carbon in the form of VFAs rather than allowing its conversion into methane. Third, DES-based extraction can alleviate product inhibition by removing accumulated VFAs from the fermentation environment, which helps maintain a more favorable pH and reduces the toxicity of undissociated acids to microbial cells. Although extractive approaches do not directly suppress methanogenesis or alter microbial metabolic pathways, they can substantially improve VFA accumulation, process stability, and overall productivity when integrated with fermentation control strategies.

The extraction efficiency of VFAs is strongly influenced by the pH of the system, as effective liquid–liquid extraction typically requires VFAs to be in their undissociated form. This condition is generally achieved at pH values below the pKa of the corresponding acids. Conventional extractants predominantly interact with undissociated VFA molecules and therefore exhibit limited effectiveness under neutral or alkaline conditions, which are often maintained in fermentation broths to support the activity of acidogenic anaerobic microorganisms [87,88]. However, the versatile components of hydrophobic ionic DESs, such as the DES made of decanoic acid/quaternary ammonium salt [89], have the potential to attract both dissociated and undissociated forms of carboxylic acids, thereby expanding their application across various pH conditions. Additionally, the high boiling points and decomposition temperatures for DESs offer the possibility of easy separation of VFA and solvent recycling through evaporative methods [68,90].

Hydrophobic DESs (HDESs) have demonstrated potential for the extraction of VFAs from fermentation broth. Various DESs—such as those composed of dihexylthiourea and trioctylphosphine oxide [88], menthol and lauric acid [68], and decanoic acid with various quaternary ammonium salts [89]—have exhibited higher distribution coefficients, i.e., the ratio of targeted solute between the extractant phase and aqueous phase at equilibrium, for carboxylic acids at low concentrations and the ability to extract VFAs from aqueous solutions. Van Osch et al. [89] demonstrated the feasibility of using HDESs for the extraction of VFAs from aqueous solutions. In this work, ionic-based HDESs were applied to recover acetic, propionic, and butyric acids, with all tested systems exhibiting superior extraction efficiencies compared to the conventional benchmark solvent trioctylamine. Subsequent studies further expanded the scope of HDES-based extraction systems. For instance, van den Bruinhorst et al. [88] reported that mixtures of dihexylthiourea and TOPO serve as highly effective extractants for VFAs. Similarly, Rodrigues-Llorente et al. [69] demonstrated that an HDES composed of octanoic acid and thymol achieves comparable or even higher extraction efficiencies than conventional organic solvents reported in the literature. These studies also highlight that the extraction of VFAs is attributed to ion exchange and intermolecular interactions, particularly hydrogen bonding, between the DES anion and the carboxylic acids [89,90,91]. Moreover, the extraction efficiency generally increases with the alkyl chain length of the VFAs and the extraction performance could be tuned by modifying the composition of the DES components [88].

In addition, HDES can be integrated with membrane extraction system for continuous extraction of VFAs from fermentation broth. For instance, Zhang et al. [73] developed a membrane contactor system integrating an omniphobic membrane with a menthol-based hydrophobic DES (Figure 2) to achieve selective, water-free extraction of VFAs from complex digestate. High extraction efficiencies were obtained with performance increasing with carbon chain length. The process is governed by reversible adsorption and enables direct coupling with anhydrous distillation for energy-efficient product recovery. Liu et al. [92] further systematically evaluated type III and type V hydrophobic DESs for VFA extraction in both direct and membrane-assisted systems, demonstrating high recovery for longer-chain VFAs and minimal temperature dependence. Combined experimental and molecular simulation results highlight the roles of pH, temperature, and solvent structure in governing extraction behavior, providing mechanistic insight into HDES design for robust VFA recovery across varying fermentation conditions.

Following extraction, the recovery of VFAs from the DES phase and the regeneration of the solvent are critical factors in evaluating the overall feasibility and sustainability of the process. Although studies in this area remain limited, several reports have proposed viable regeneration pathways, primarily including evaporation/distillation and back-extraction. For example, Darwish et al. [68] regenerated a menthol–lauric acid HDES via rotary evaporation (303.2 K, 20 mbar, 24 h), with the recovered solvent maintaining comparable extraction performance for at least one reuse cycle. Direct distillation also represents a feasible strategy for VFA recovery and DES recycling, owing to the significant volatility differences between VFAs and DES components. Zhang et al. [73] demonstrated that VFAs are not chemically bound within the HDES matrix and can therefore be selectively recovered across different temperature ranges in a distillation column. Alternatively, back-extraction offers an effective route for VFA recovery from the DES phase by converting VFAs from their undissociated to dissociated forms using alkaline agents, thereby enhancing their affinity for the aqueous phase. Vidal et al. [94] systematically evaluated various alkaline agents (NH₄⁺, NaOH, and Na₂CO₃) and operating parameters, identifying 0.1 M NaOH as the most effective system. Their results further showed that three sequential back-extraction stages, each using fresh alkaline solution, were required to maximize acid recovery and regenerate the DES. Notably, the recycled solvent exhibited extraction performance comparable to that of the fresh DES, indicating good reusability.

Despite these promising results, several limitations remain for large-scale implementation of DES- and HDES-based extraction systems. Although DESs are frequently described as green and biocompatible solvents, most current studies have evaluated their performance using purified enzyme systems or simplified laboratory conditions. Their long-term effects on complex mixed microbial consortia under realistic fermentation conditions remain insufficiently understood and require further investigation. In particular, the economic feasibility of some hydrophobic DES formulations may be constrained by the cost and availability of specific solvent components, while high solvent viscosity may negatively affect mass transfer and phase separation during continuous operation [95,96]. In addition, regeneration strategies involving alkaline back-extraction or multi-stage solvent recovery may increase chemical consumption and downstream processing complexity. Although many DES systems have demonstrated favorable recyclability at the laboratory scale, further long-term studies are still needed to evaluate solvent stability, process economics, and industrial sustainability under realistic fermentation conditions.

Beyond direct extraction and recovery, downstream upgrading of VFAs into higher-value products provides additional opportunities for process intensification and product valorization. In particular, esterification can improve product separability, reduce acid toxicity, and generate value-added compounds with applications in solvents, flavors, fragrances, plasticizers, and biofuels [97,98]. Therefore, integrating VFA extraction with esterification represents a promising complementary strategy for enhanced VFA utilization. Notably, pH plays mechanistically distinct roles in extraction (favoring acid protonation for partitioning into hydrophobic phases) and esterification (often requiring acid catalysis at low pH), creating an inherent compatibility tension for integrated single-vessel configurations that must be carefully balanced in process design. The use of DES in conjunction with lipases can partially bridge this gap, as appropriately tailored DES can provide a biocompatible microenvironment that stabilizes lipase and supports catalytic activity at moderate pH, enabling simultaneous extraction and conversion without imposing harsh acidic conditions.

5. VFA Esterification

DES can play a versatile role in esterification reactions, serving as a solvent, substrate reservoir, catalyst, catalyst booster, or performing combined functions for VFA esterification (Figure 3). For example, DESs composed of quaternary ammonium methanesulfonate salts and p-toluenesulfonic acid (PTSA) could act as efficient dual solvent-catalysts for esterification of carboxylic acids with alcohols, where they exhibit high activity, easy recovery, and reusability while operating under mild conditions of 60~80 °C [99]. Two DESs based on triphenylphosphonium bromide/PTSA monohydrate and ChCl/Gly were discovered to enhance the esterification process of fatty acids in low-grade oil, where the former acted as a catalyst [100], while the latter served as a catalyst booster for the Amberlyst catalyst [101]. Both DESs demonstrated promising reusability potential, with catalysts being recycled for 4 or 5 cycles while maintaining a high conversion rate of over 90% for fatty acids. In other cases, the HBD of ChCl-based DESs was found to act as a substrate in esterification reactions such as carboxylic acid [102] and quaternary ammonium salt (as an alkylating reagent) [103]. It was also reported that esterification substrates racemic menthol, and lauric acid could be prepared into a DES as the solvent and facilitate their reaction to produce menthyl laurate [104].

In addition, certain DESs have shown potential as suitable media for enzyme-catalyzed esterification. Lipases, known for their versatility and robustness in diverse reactions, have been extensively studied in DES systems. Recent papers have reviewed the lipase species, their catalyzed reactions, and their performance in different DESs [29,105,106]. Remarkably, lipases have exhibited advantageous characteristics, with the retention or even enhancement of activity and stability in specific DESs, such as those containing ChCl or glycerol [29,105]. For the lipase-catalyzed esterification reactions in DESs, DESs served not only as an effective solvent for substrates with different polarities [107] but sometimes as a reservoir of the substrate as well, enabling a higher substrate loading and conversion rate [104,107,108,109,110]. The acids employed in these reactions are typically characterized by lower polarity, such as benzoic acid [107,109], n-3 polyunsaturated fatty acids [111], and other long-chain fatty acids (with a carbon number no less than 8) [108,110,112], among others [106]. However, lipase-catalyzed esterification of the polar VFAs produced from acidic fermentation in a DES system is underexplored. Integration of VFA extraction and esterification in DESs could offer a unique process intensification opportunity for improved productivity of the whole VFA production, recovery, and valorization system.

Although lipases are among the most solvent-tolerant biocatalysts, DES environments can still perturb enzyme conformation and hydration layers, leading to reduced activity or stability. Enzyme engineering and functionalization are essential to enable robust lipase catalysis in DES systems, where enzyme performance is governed by solvent polarity, hydrogen-bonding interactions, and water activity [113]. To mitigate deactivation, protein-level modifications such as surface charge engineering and enzyme–polymer conjugation have been explored to enhance structural rigidity and solvent compatibility [114]. Among these strategies, enzyme immobilization remains the most effective and widely applied approach. Immobilization methods—including adsorption on hydrophobic carriers, covalent attachment, and cross-linked enzyme aggregates (CLEAs)—can create protective microenvironments that preserve enzyme structure, maintain essential hydration, and improve resistance to solvent-induced denaturation [115,116].

Recent advances demonstrate that immobilized lipases—particularly Candida antarctica lipase B (CALB)—can retain substantial catalytic activity in DES-based systems, enabling efficient esterification and transesterification under mild conditions [117]. The compatibility between lipases and DESs is highly system-dependent, with hydrophobic or low-polarity DESs generally providing more favorable environments by minimizing disruption to the enzyme’s hydration shell. In addition, emerging DES formulations, including NADES and reactive DESs, have been shown to enhance enzyme activity and stability while simultaneously participating in catalytic processes, thereby improving reaction efficiency. Advanced immobilization designs—such as confinement within hydrophobic matrices or hierarchical porous supports—further improve enzyme–solvent compatibility and mass transfer characteristics.

In a recent study, Liu and Shi [56] investigated nanoconfined lipase biocatalysts for in situ VFA esterification, achieving >75% enzyme retention and >97% stability for Aspergillus oryzae lipase under acidic conditions, while Candida rugosa lipase exhibited ~20-fold higher activity. The system showed improved esterification selectivity at 25 °C, although its catalytic activity was 1–2 orders of magnitude lower than free enzymes due to mass transfer limitations. DESs can enhance VFA esterification by serving as a compatible reaction medium that enables simultaneous extraction and lipase-catalyzed conversion under mild conditions. Hydrophilic DESs such as choline chloride/ethylene glycol (1:2) and choline chloride/levulinic acid (1:2) have been shown to maintain or even enhance lipase activity, thereby promoting efficient biocatalytic ester formation, whereas certain hydrophobic DESs may inhibit enzyme performance [118]. Collectively, these enzyme-engineering strategies provide opportunities for improving biocatalysis efficiency in integrated DES-based extraction and esterification systems and represent a key enabling technology for advancing VFA valorization processes.

6. Perspectives and Conclusions

AAD represents a promising platform for converting organic waste into value-added VFAs. Two critical levers for improving VFA production are feedstock pretreatment and process control. Pretreatment can effectively disrupt the recalcitrant lignocellulosic structure, increase substrate accessibility, and accelerate hydrolysis and acidogenesis. Concurrently, strategies that suppress methanogenesis and maintain favorable fermentation conditions—such as low pH, thermophilic temperature, high organic loading, and short hydraulic retention time—are essential to direct carbon flux toward VFA accumulation rather than methane formation.

Despite improvements in upstream processing, the accumulation of VFAs introduces product inhibition that can suppress microbial activity and limit overall productivity. Addressing this constraint requires the integration of in situ product removal strategies. Technologies such as membrane separation, adsorption, and liquid–liquid extraction can continuously remove VFAs from the fermentation broth, thereby stabilizing the microbial environment and enabling sustained production. Among these approaches, hydrophobic DESs have emerged as particularly promising extractants due to their tunable physicochemical properties, low volatility, and potential biocompatibility. Notably, certain hydrophobic natural DESs can selectively extract VFAs even in their dissociated forms under neutral or mildly alkaline conditions, offering operational flexibility and improved separation performance. However, further investigation is needed to evaluate their performance in real fermentation systems and to integrate them with complementary separation technologies.

Beyond extraction, coupling VFA recovery with downstream valorization is critical for improving process economics and overall sustainability. In situ esterification represents a compelling strategy to simultaneously mitigate acid inhibition and convert VFAs into higher-value ester products with improved separability. Hydrophobic DESs play a multifunctional role in this context, acting not only as extraction media but also as solvents, catalysts, or co-catalysts for esterification reactions. Recent studies have demonstrated that lipases—both free and immobilized—can retain activity in DES-containing systems, enabling the design of biphasic aqueous–DES systems for simultaneous extraction and catalytic upgrading. In particular, immobilized lipases confined within hydrophobic microenvironments have shown promise for enhancing reaction stability and selectivity. Nevertheless, systematic investigations into aqueous-phase esterification and the integration of extraction–reaction systems remain limited and warrant further research. Despite these developments, a predictive understanding of enzyme behavior in DES environments remains limited. Increasing attention is therefore being directed toward molecular-level approaches, particularly molecular dynamics simulations, to elucidate solvent–enzyme interactions and guide the rational design of DES-compatible biocatalytic systems for integrated extraction and in situ esterification processes.

From a systems perspective, process intensification provides a unifying framework for integrating these upstream and downstream strategies into efficient and scalable platforms. By combining feedstock pretreatment, controlled fermentation, in situ VFA removal, and biocatalytic upgrading within a single process architecture, process intensification can reduce intermediate handling, improve mass and heat transfer, and enhance overall process efficiency. However, several challenges must be addressed to enable industrial implementation. Techno-economic analysis is required to assess the cost-effectiveness of integrated systems, particularly considering the use of DESs and advanced biocatalysts. In addition, molecular-level understanding of DES–enzyme interactions remain limited. While docking simulations offer preliminary insights, MD simulations provide a more robust approach for capturing solvent–enzyme–substrate interactions and guiding the rational design of compatible systems. Advancing these modeling capabilities, alongside experimental validation, will be essential for optimizing integrated VFA production, recovery, and valorization processes and realizing their potential in sustainable biorefinery applications.

Although DES- and HDES-based recovery systems have demonstrated promising extraction performance at laboratory scale, significant challenges remain for pilot-scale and industrial implementation. Future studies should further evaluate continuous operation, solvent regeneration efficiency, membrane stability, and process robustness under industrially relevant fermentation conditions. In addition, pilot-scale validation and techno-economic analysis will be important for assessing the practical feasibility of integrated DES-enabled VFA biorefineries.

In conclusion, the integration of AAD, feedstock pretreatment, in situ VFA removal, and biocatalytic upgrading through esterification represents a compelling pathway for advancing VFA-centered biorefineries. Hydrophobic DESs, in particular, offer a versatile platform for coupling extraction and reaction processes, enabling simultaneous recovery and valorization of VFAs while mitigating product inhibition. However, significant challenges remain in process integration, system optimization, and economic viability. Addressing these gaps through interdisciplinary approaches—combining process intensification, advanced separation technologies, biocatalyst engineering, and molecular-level modeling—will be critical to translating these concepts into scalable and sustainable industrial applications.