# Advancing sustainable closed-loop solutions: Supercritical carbon dioxide technology for aquatic biological waste valorization

Qiqi Sun, Jean-Christophe P. Gabriel, Lay Poh Tan

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Qiqi Sun, Jean-Christophe P. Gabriel, Lay Poh Tan. Advancing sustainable closed-loop solutions: Supercritical carbon dioxide technology for aquatic biological waste valorization. Applied Food Research, 2025, 5 (2), pp.101384. 10.1016/j.afres.2025.101384. cea-05290309

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# Advancing sustainable closed-loop solutions: Supercritical carbon dioxide technology for aquatic biological waste valorization

Qiqi Sun<sup>a, b, c, e</sup>, Jean-Christophe P. Gabriel<sup>b, c, e</sup>, Lay Poh Tan<sup>a, e</sup>

## ARTICLE INFO

### Keywords:

Supercritical CO₂

Aquatic waste

Sustainable recycling

## ABSTRACT

The issue of aquatic food waste has drawn significant attention. As a waste rich in valuable nutrients, reusing it can protect the environment while generating economic benefits. Supercritical, subcritical liquid, and gaseous CO₂ treatments have the advantages of being non-toxic, easy to recycle and not producing harmful byproducts. This review summarizes the utilization of supercritical carbon dioxide (ScCO₂) for the reuse of aquatic waste, including fish and algae, through extraction and fractionation to obtain omega-3, phospholipids, carotenoids, collagen, and other bioactive compounds, as well as its combination with other technologies to enhance efficiency. Additionally, it discusses the thermodynamic and kinetic modeling of the processes and their economic feasibility. Given the advantages of ScCO₂ technology, it has the potential to see broader adoption in the aquatic food industry.

## 1. Introduction

In recent years, the application of ScCO₂ technology in food waste treatment has garnered increased attention. A search using the keywords "supercritical carbon dioxide" and "food waste" in Web of Science yielded a total of 158 articles published from 2021 to 2025 (Fig. 1).

At the beginning of 1990s, the concept of Green Chemistry was proposed. Solvents often account for the majority of the mass waste produced in processes and are therefore an important challenge for Green Chemistry (Santos et al., 2022). Many organic solvents are toxic, flammable, corrosive, and volatile, which potentially lead to air, water, and land pollution. ScCO₂ offers a new green solution as it is a sustainable gas considered safe by the U.S. Food and Drug Administration (FDA) (Amani et al., 2022; Luca et al., 2023).

Furthermore, ScCO₂ regeneration is simple compared to that of organic solvents, which requires energy-intensive steps such as distillation, making the classical separation process one of the most energy-consuming steps in fractionation (Satari & Jaiswal, 2021). For ScCO₂, all that is required is to change the pressure or the temperature. When CO₂ is below the critical point, it becomes a gas, and degassing the system removes the solvent completely (Fig. 2). In addition, the post-process recovery of CO₂ enables its recycling and improves

economic efficiency (Paterson et al., 2024).

ScCO₂, a medium for carbon capture, promotes carbon reuse, and this enables of innovative ways for CO₂ recycling and carbon emission reduction, hence it has the potential to be an important technology asset for achieving carbon neutrality and sustainable development goals (Gür, 2022). The Global Carbon Project (GCP) has released a global CO₂ emissions budget report for 2024, pointing out that global CO₂ emissions will once again hit a record high, with emissions expected to reach 37.4 billion tons, an increase of 0.8% over 2023. In addition, the introduction of economy-wide decarbonization targets by 2050 has also stimulated carbon capture, utilization, and storage (CCUS) in industrial applications (Rode et al., 2023).

One of the advantages of ScCO₂ treatment is that it generates no by-products, leaving no residue and yielding a high-value extract from the initial raw material (Alassali et al., 2020). Its low chemical reactivity renders CO₂ inert and typically does not oxidize substrates or products, and it is both non-toxic and non-mutagenic (Perva-Uzunalić et al., 2004; Traversier, Gaslonde, Lecso et al., 2020) (Dal Prá et al., 2013). It also allows for low-temperatures operations, thus minimizing organic decomposition or denaturation (Deng, Xia, Bourgeois et al., 2024). As a result, it has found extensive applications in the food industry, particularly in extraction, sterilization, preservation, and the removal of

<footnote><sup>a</sup> School of Materials Science and Engineering, Nanyang Technological University, 639798, Singapore</footnote>

<footnote><sup>b</sup> SCARCE Laboratory, Energy Research Institute @ NTU (ERI@N), Nanyang Technological University, Singapore, 637459, Singapore</footnote>

<footnote><sup>c</sup> LICSEN, NIMBE, CNRS, CEA, Université Paris-Saclay, Gif-sur-Yvette, 91191, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: jean-christophe.gabriel@cea.fr (J.-C.P. Gabriel), LPTan@ntu.edu.sg (L.P. Tan).</footnote>
harmful substances. (Mukhopadhyay & Chatterjee, 2023). Despite the
advantages of ScCO₂, it has certain limitations, one of which is the high
equipment investment required for high pressure, along with the asso-
ciated risks of operating at such pressures. Additionally, ScCO₂ extrac-
tion without co-solvent may be less effective for extracting polar
compounds (Duba & Fiori, 2019).

Aquatic foods, meanwhile, are one of the most traded food com-
modities in the world. The United Nations Food and Agriculture Orga-
nization (FAO) released the State of World Fisheries and Aquaculture
2024 and noted that global fisheries and aquaculture production surged
to 223.2 million tons in 2022, an increase of 4.4 % over 2020 (FAO,
2024). However, the loss and waste of aquatic food is a major challenge
to the sustainable development of the market representing a loss and
waste of aquatic food of about 22.7 % in the United States (Love et al.,
2023). Among them, fish by-products, including fish skin, fish head, fish
guts, fish bones, etc., are potentially important nutritional sources of
protein, fatty acids, and minerals. To date, fish waste has primarily been
utilized in the fishmeal industry (Coppola et al., 2021). Instead, fish
waste could be further exploited, for example, by extracting
omega-3-rich fish oil and producing high-value products that promote
sustainable growth (Caruso et al., 2020). Crustacean by-products, that
are mainly composed of exoskeletons, can be used to extract chitin
Azelee et al., (2023). Hence, a proper treatment of aquatic food waste
could both help protect the environment and yield economic benefits.

The applications of ScCO₂ for aquatic waste treatment include pro-
cessing methods such as extraction, fractionation, and drying, as well as
preservation processes like enzyme deactivation and sterilization
(Barbosa et al., 2020; Bernardo et al., 2024). In this work,
state-of-the-art ScCO₂ extraction and fractionation processes of fish
waste are critically reviewed.

2. ScCO₂ extraction in aquatic waste extraction

Under supercritical conditions, CO₂ properties can be tuned to have a
gas-like viscosity and a liquid-like density, which enhances solubility
and flow characteristics (Kravanja et al., 2022). It can selectively extract
non-polar compounds, but by adding co-solvent, the solubility and
selectivity can be significantly changed (Sahena, Zaidul, Jinap et al.,
2009)(Deviese, Van Ham-Meert, Hare et al., 2018). ScCO₂ extraction
consists of two fundamental stages: dissolution and diffusion. Initially,
ScCO₂ penetrates, diffuses into the solid matrix, and dissolves the solute,
which then undergoes a two successive transport steps: (1) diffuses
through the matrix to the outer layer, and (2) transfer from the surface
into the bulk solution (Xia, Maurice, Leybros et al., 2021). The three
main parameters that control the ScCO₂ extraction are pressure, tem-
perature, and the extraction time. Under isothermal conditions, an in-
crease in pressure generally enhances the solvent power of ScCO₂ due to
its elevated density. Conversely, under isobaric conditions, raising the
temperature leads to an increase in the vapor pressure of the solute,
which favors extraction; however, it also decreases the density of CO₂,
potentially reducing its solvating capacity. In addition, the use of
co-solvent, CO₂ flow rate, the pretreatment of the raw material,
including moisture content, particle size, etc. also affected the extraction
process (Badgujar et al., 2021; Coelho et al., 2020; Kravanja et al.,
2022). In the work by Charest et al. (Charest, Balaban, Marshall et al.,
2001), they pointed out that moisture content and the presence of a
co-solvent significantly influenced the extraction behavior. The authors
observed a nonlinear effect of moisture content on astaxanthin extrac-
tion, with the highest yield occurring at an intermediate moisture level
(approximately 13 %–41 %). However, some studies have reported that
the amount of oil extracted increases significantly as moisture content
decreases, as the presence of water reduces the solubility of oil in ScCO₂
(Dunforda et al., 1998; Fornari, Vázquez, Villanueva-Bermejo et al.,

<figure><img src="image_3.png" /><figcaption>Fig. 1. Co-citation network visualization of the "ScCO₂" and "Food waste" publications. Citation relationships for 2021–2025 (VOSviewer).</figcaption></figure>
2023). Similarly, the use of co-solvents should be selected based on the specific property of compounds, as it may increase the product yield (Getachew et al., 2024) but could also reduce the separation efficiency of the target compounds (Milovanovic, Grzegorczyk, Świątek et al., 2025). Due to differences in raw materials and target compounds, the key parameters affecting the extraction process vary significantly. In the following sections, the common extracts are discussed.

## 2.1. Different valuable bioactive compounds

Fig. 3 below shows the chemical structures of some common bioactive compounds and lipids that could be extracted using ScCO₂. These extracts will be discussed in greater detail subsequently.

### 2.1.1. Lipids

#### 2.1.1.1. Omega-3
Omega-3 fatty acids (See Fig. 3) are a type of unsaturated fatty acid that contains C = C double bonds, with the first double bond located at the third carbon atom from the methyl end (omega) of the fatty acid chain. Omega-3 fatty acids include three essential fatty acids that are beneficial to human health: alpha-linolenic acid (ALA), eicosatetraenoic acid (EPA), and docosahexaenoic acid (DHA), which are represented in Fig. 3A-C, respectively (Tocher, 2015). These fatty acids cannot be synthesized from available nutrients within the human body and must therefore be obtained through dietary sources. Although ALA can be converted into EPA, the conversion rate is low, making direct intake from food a more convenient option (Liao et al., 2022). Scientific research has shown that EPA and DHA have positive impacts on health, including reducing the risk of cardiovascular diseases, cancer, and diabetes, lowering the chance of depression, and supporting the immune system while ensuring proper neural development (Swanson et al., 2012).

Marine fish and algae can both provide omega-3. Fish oil is a primary natural source of long-chain Omega-3 fatty acids containing EPA and DHA (Herrera et al., 2013). Historically, fish oil has played a significant role in human diets, and with growing evidence of its benefits, the

demand for fish oil continues to rise. Fish oil is primarily used in the food and pharmaceutical industries, as well as in agriculture and aquaculture as a feed additive (Vaisali et al., 2015). Given the benefits of fish oils, extraction methods have been widely explored both in research and industry. The most common method for producing fish oil is the wet reduction process, which involves three key steps: high-temperature cooking (85–95 °C), pressing, and centrifugation (Taati et al., 2018). While common, the main disadvantages of this method are that high temperature can release lots of heat and require further refining steps to enhance the quality of oil (Vaisali et al., 2015). In this review, a greener method using ScCO₂ is explored.

**Table 1** summarizes the yields, advantages, and limitations of ScCO₂ extraction using different fish species under various experimental conditions. The yield of fish oil depends on the fish body part (Sahena et al., 2010) and the experimental parameters of ScCO₂ (Shalfoh et al., 2024). This information helps in understanding the effectiveness of different process protocols for utilizing fish by-products. The table shows that when extraction is carried out at 40–60 °C and 15–30 MPa, most studies achieve relatively high oil recovery yields. At a given temperature, an increase in pressure leads to a significant increase in yield, whereas at a constant pressure, raising the temperature does not markedly affect the yield. Additionally, higher temperatures may increase extraction efficiency, but they also bring higher energy consumption and a risk of lipid oxidation. The CO₂ flow rate affects the time required to reach the maximum extraction yield. A higher flow rate leads to faster extraction, but the final yield shows little difference. However, the CO₂ utilization efficiency is lower at higher flow rates. Conversely, a very low CO₂ flow rate result in prolonged extraction times, making it unsuitable for industrial applications. Therefore, optimization should fully consider energy efficiency, balancing both extraction efficiency and processing time.

In addition, it is also observed that current research is largely confined to laboratory-scale studies, lacking systematic economic evaluations and examples of large-scale continuous production. Future research should include assessments of economic feasibility.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** CO₂ Phase Diagrams.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 3. Molecular Structures of Major Bioactive Compounds.</figcaption></figure>

### 2.1.1.2. Phospholipids.
ScCO₂ has been used to extract phospholipids from Pleuroncodes monodon(Barriga-Sánchez et al., 2023), Greenshell mussel meat (Morel et al., 2024), Atlantic shrimp (Ahmadkelayeh et al., 2022), Conger eel (Park et al., 2024), which is shown in Fig. 3D, (Barriga-Sánchez et al., 2023)(Morel et al., 2024)(Ahmadkelayeh et al., 2022)(Park et al., 2024), etc. Phospholipids are a type of polar lipid, and polar lipids have relatively low solubility in ScCO₂. The solubility of phospholipids in ScCO₂ is limited at pressures below 50 MPa. To enhance their solubility, methanol or ethanol is commonly used as co-solvents to modify the polarity of ScCO₂ (Zhou & Li, 2009). Han et al. (Han et al., 2024) found that short-chain alcohols (e.g., MeOH) form stronger hydrogen bonds with the polar head of phospholipids, enhancing solubility in ScCO₂. MeOH creates a high-density solvation shell, boosting ScCO₂'s solvent strength and optimizing extraction efficiency, making it the most effective entrainer. In addition, the extraction rate of phospholipid could also be improved by increasing the proportion of co-solvent in a certain range. Savoire et al. (Savoire et al., 2020) increased phospholipid extraction from 15.9 % to 81.2 % by raising ethanol from 7 % to 30 %.

Haq et al. (Haq & Chun, 2018) compared the extraction of phospholipids from salmon frame bone using ScCO₂ with ethanol as a co-solvent and ethanol alone. They found that the ScCO₂ group yielded phospholipids with higher purity and richer content of astaxanthin and omega-3 PUFAs. Additionally, phospholipids extracted using this method exhibited better antioxidant activity, emulsification properties, and thermal stability. Nolwenn et al. (Terme, Boulho, Kucma et al., 2018) also believe phospholipids extracted using ScCO₂ exhibited higher antioxidant activity (DPPH radical scavenging rate) compared to those obtained by conventional chloroform/methanol extraction. However,

the authors noted that the yield of phospholipids from ScCO₂ extraction was slightly lower than that of conventional ethanol extraction, which might be due to ScCO₂ primarily extracting non-polar components, leaving some phospholipids in the residue (Zhou & Li, 2009) (Yuqing Han et al., 2024) (Savoire et al., 2020).

From a mechanistic point of view, the solubility of phospholipids in ScCO₂ largely depends on intermolecular hydrogen bonding. Based on the composition analysis of extracts, choline-based phospholipids (such as phosphatidylcholine and sphingomyelins, as shown in Fig. 4) have the highest solubility, which correlates well with their head groups having moderate polarity and therefore a good affinity with ScCO₂. The choline group is shown in the grey part of Fig. 3D In contrast, serine- and inositol-based phospholipids are almost insoluble due to their highly polar head groups and strong hydrogen bonding interactions (Güçlü-Üstündağ & Temelli, 2005). The solubility of phospholipids can be enhanced through their head chemical modification, using molecules such as polyvinyl acetate (PVAc), which reduces intermolecular hydrogen bonding (Song et al., 2020; Yuexia Han, Cheng, Ruan & Jiao, 2021).

To summarize, co-solvents or chemical modifications are required for effective ScCO₂ extraction of phospholipids, due to their low solubility and strong hydrogen bonding interactions. While co-solvent improves extraction in general, the main disadvantage of using a co-solvent is post-process solvent residue, which requires additional procedures for removal, thus increasing costs.

### 2.1.1.3. Carotenoids.
Carotenoids are natural lipophilic pigments, including β-carotene, lutein, and astaxanthin Manzoor et al., Gani and Noor (2021). Oxidative stress is related to the development of some
<table><caption>**Table 1**
Overview of ScCO₂ most recent methods for oil extraction (2021–2025).</caption><thead><tr><th>Species</th><th>Experimental parameter</th><th>Co-solvent</th><th>Oil yield</th><th>Highlights</th><th>Limitations</th><th>Reference</th></tr></thead><tbody><tr><td>Sardines</td><td>35–75 °C, 300–550 bar, CO₂ flow rate 5–25 g/min</td><td>None</td><td>20.93 %- 25.59 %</td><td>Antiproliferative and anti-inflammatory effects on cancer cell lines</td><td>Lower oil yield compared to B&amp;D extraction; slower extraction at lower CO₂ flow</td><td>(Rodrigues et al., 2021)</td></tr><tr><td>Catfish (Netuma barba)</td><td>40–60 °C, 15–25 MPa, time 1–3 h</td><td>None</td><td>83.8 % of B&amp;D</td><td>Comparable to refined thermomechanical oil; refining not required</td><td>EPA and DHA lower than thermomechanical product</td><td>(da Silva, Igansi, da Silva et al., 2023)</td></tr><tr><td>Yellowtail (Seriola quinqueradiata)</td><td>40–50 °C, 15–30 MPa, CO₂ flow 27 g/min</td><td>None</td><td>40.87 % (ScCO₂), 48.48 % (n-hexane)</td><td>High omega-3 fatty acids, DHA and EPA; high oxidative stability in oil</td><td>Lower yield compared to solvent-based extractions</td><td>(Franklin et al., 2020)</td></tr><tr><td>Cucumaria frondosa (C. frondosa)</td><td>35–80 °C, 20–50 MPa, time 30–70 min</td><td>Ethanol</td><td>16.30 ± 0.66 g/100 g of dried sample</td><td>Freeze-Drying and Ethanol-Soaking Enhance Yields; Low omega-6/omega-3 ratio</td><td>Lower DHA Content; limited Suitability for Polar Compounds</td><td>(Lin, Jiao, Brooks et al., 2024)</td></tr><tr><td>Arctic Charr (Salvelinus alpinus)</td><td>40–80 °C, 20–45 MPa, CO₂ flow 30 g/min</td><td>None</td><td>15.4 %- 35.4 %</td><td>Rich in PUFA and astaxanthin; sustainable extraction method for fish oil production</td><td>Oxidation issues at higher temperatures</td><td>(Semenoglou et al., 2021)</td></tr><tr><td>Jack mackerel (WJH); Hoki (HFH); Greenshell mussel (GSM)</td><td>40 °C, 300 bar</td><td>None</td><td>16.7 g/100 g dried sample (WJH); 12.3 g/100 g dried sample (HFH); 5.9 g/100 g sample (GSM)</td><td>High-quality natural lipids (especially omega-3)</td><td>Insufficient of polar lipid extraction capacity</td><td>(Liu, Castagnini, Berrada et al., 2025)</td></tr><tr><td>Anchovy</td><td>55 °C, 30 MPa</td><td>None</td><td>15.1 % (ScCO₂), 19.9 % (Hexane)</td><td>High in omega-3 PUFA and vitamins A, D, E; ScCO₂ method produces purer oil</td><td>Higher peroxide value compared to commercial oils, requiring further refinement</td><td>(J. S. Park et al., 2021)</td></tr><tr><td>Starfish (Asterias rubens)</td><td>45 °C, 275 bar, ethonal flow rate 1–3mL/min</td><td>Ethanol</td><td>About 50 % of B&amp;D</td><td>High purity</td><td>Low yield; need to optimize conditions</td><td>(Sørensen et al., 2022)</td></tr><tr><td>Starfish (Asterias rubens)</td><td>40–50 °C, 250–350 bar, CO₂ flow rate 3–12 mL/min</td><td>Ethanol</td><td>14.56–21.16 %</td><td>High in phospholipid, α-tocopherol, EPA and DHA</td><td>Initial peroxide value is high, indicating improper pretreatment process</td><td>(Getachew et al., 2024)</td></tr><tr><td>Eel (Conger myriaster)</td><td>55 °C, 30 MPa, CO₂ flow 27 g/min</td><td>None</td><td>NA</td><td>High brightness; low heavy metal content.</td><td>Lower α-tocopherol content than cold-pressed oil</td><td>(Park, Roy, Kim et al., 2022)</td></tr><tr><td>Cod (Liver)</td><td>49 °C, 29.9 MPa, CO₂ flow 4.97 mL/min</td><td>None</td><td>17 %, rich in omega-3 PUFAs</td><td>High in squalene, encapsulated to improve stability and antioxidant properties</td><td>Encapsulation process adds cost and complexity</td><td>(Lee, Surendhiran, &amp; Chun, 2022)</td></tr><tr><td>Skipjack Tuna (Katsuwonus pelamis)</td><td>55 °C, 30 MPa, CO₂ flow 27 g/min; 40 °C, 30 MPa, CO₂ flow 27 g/min, 10 % co-solvent</td><td>Ethanol</td><td>31.18 % (neutral lipids), 3.24 % phospholipids</td><td>High PUFA content (EPA, DHA), phospholipids extracted from head, tocopherol detected in lipids</td><td>Requires further research on in-vitro and in-vivo potentialities</td><td>(Shin et al., 2024)</td></tr><tr><td>Japanese Spanish Mackerel (Scomberomorus niphonius)</td><td>45 °C, 250 bar, 3 h</td><td>None</td><td>42.79 % skin, 31.08 % head, 22.70 % viscera</td><td>High EPA (5.81 %) and DHA (12.56 %) in skin, low FFA, good antioxidant activity, thermally stable</td><td>Lower yields compared to Soxhlet extraction due to selective extraction</td><td>(Roy et al., 2022)</td></tr></tbody></table>

chronic diseases. Carotenoids, as a kind of antioxidants, have gained attention for their potential role in mitigating this process. Moreover, carotenoids provide color and play an important role in the food industry as natural pigments. The general structural backbone of carotenoids is illustrated in prior section in Fig. 3, while specific end-group structures (R groups) of these representative molecules commonly extracted by ScCO₂ are summarized in Table 2.

A main issue regarding their extraction is that their highly conjugated system makes them susceptible to oxidation, which is what makes them great antioxidant molecules (Lako et al., 2007). Hence, if either oxygen, light, or heat is present, carotenoids may be subject to oxidative degradation. This degradation not only occurs during the extraction process but can also continue during heat treatment, light exposure, or storage. Fig. 5 shows a typical oxidative degradation process of β-carotene (Koschmieder et al., 2021). Once oxidation is initiated, carotenoids may further react with other substances, forming various low-molecular-weight products. Overall, oxidative degradation has been reported to be the primary cause of extraction loss (Aslam et al., 2021). The non-solvent ScCO₂ can slow down such degradation and therefore preserve their antioxidant activity (Mattea et al., 2009).

Although the non-polar nature of carotenoids theoretically allows for extraction using pure ScCO₂, their high molecular weight limits the extraction yield without a co-solvent (Ndayishimiye & Chun, 2017). The use of a co-solvent effectively enhances the extraction yield from 34.9 %

to about 82.5 % when 18.4 % ethanol was used (de Andrade Lima et al., 2018). Sovová et al. (Helena Sovová & Stateva, 2019) described the extraction of carotenoids as their dissolution in microalgal lipids and found that the solubility of carotenoids is positively correlated with that of triglycerides. They further demonstrated that incorporating edible oils (such as triolein) as co-solvents can enhance the solubility of carotenoids. The extraction rate is mainly controlled by solubility and adsorption capacity; optimizing extraction conditions (such as pressure and temperature) or reducing the adsorption of carotenoids by the matrix can effectively improve the extraction yield. Both pressure and temperature are critical parameters; however, pressure exhibits a markedly higher influence on carotenoid extraction yield, as evidenced by a p-value of 0.0010 compared to 0.3288 for temperature (Hosseini et al., 2017). Increasing the CO₂ flow rate within a certain range can shorten the extraction time and improve production efficiency, but it does not seem to significantly increase the final extraction yield (Espinosa Alvarez et al., 2020).

While ScCO₂ typically requires relatively high pressures and temperatures, subcritical fluid extraction offers a milder alternative with propane (Daood et al., 2002), dimethyl ether (Goto et al., 2015), carbon dioxide (Rutkowska & Stolyhwo, 2009), etc. being suitable extraction solvents. Jaime et al. (Jaime et al., 2007) used supercritical and subcritical carbon dioxide to extract carotenoids and found that high pressure (437 atm) combined with low temperature (9.8 °C), which falls
<figure><img src="image_6.png" /><figcaption>(A) Phosphatidylcholine</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>(B) Sphingomyelins</figcaption></figure>

<caption>Fig. 4. Molecular structures of phosphatidylcholine and sphingomyelins.</caption>

<table><caption>**Table 2**
Representative carotenoids commonly extracted using ScCO₂.</caption><thead><tr><th>Representative carotenoid</th><th>End group (R)</th><th>Structure of End group (R)</th></tr></thead><tbody><tr><td>β-Carotene</td><td>β-ring</td><td></td></tr><tr><td>Lutein</td><td>β-ring + ε-ring</td><td></td></tr><tr><td>Astaxanthin</td><td>β-ring (hydroxyl and keto substituted)</td><td></td></tr></tbody></table>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

within the subcritical/near-supercritical region, resulted in the highest extraction yield (6.72 %). Extracts obtained under subcritical conditions exhibited higher antioxidant activity, with a higher content of 9-cis-β-carotene and a reduced proportion of the all-trans isomer, whereas the opposite trend was observed under supercritical conditions.

Overall, carotenoids hold great market potential in the food, pharmaceutical, and cosmetics industries, due to their highly efficient antioxidant properties, extensive health benefits, and diverse industrial applications (Razz, 2024). Compared to traditional methods, supercritical/subcritical carbon dioxide extraction of carotenoids offers significant advantages in terms of extraction efficiency, environmental

sustainability, bioactivity preservation, selectivity, and industrial adaptability.

### 2.1.2. Collagen/Gelatin

Collagen is the major structural component of the extracellular matrix (ECM) in both connective and parenchymal interstitial tissues. It exhibits great biocompatibility and biodegradability. Collagen and gelatin (denatured collagen) are widely used in the food, medical, and cosmetic industries, among others. Due to religious restrictions, the acceptance of porcine and bovine gelatin is reduced. It could be replaced by collagen derived from fish skin, fish bones, and fish scales, which has
<figure><img src="image_12.png" /><figcaption>Fig. 5. Oxidation production of β-carotene.</figcaption></figure>

properties similar to porcine gelatin but with higher absorption capacity (Usman et al., 2022). The principle of collagen/gelatin extraction using CO₂ involves first to dissolve CO₂ in water by applying pressure, thus forming carbonic acidified water, which creates a mildly acidic environment that replaces traditional organic acids in the extraction process. The advantage of this method lies in its mild operating conditions and environmental friendliness. CO₂ not only has strong solubility in water but also acts as an acidifying agent under appropriate pressure and temperature, helping maintain the natural triple-helix structure of collagen during its extraction (Sousa, Martins, Carvalho et al., 2020). Unlike organic solvents, CO₂ can be released in the environment during post-processing steps, avoiding harmful chemical residues. It was also observed that the pressure reduces the odors of the ingredients (Cao et al., 2022). Therefore, Sousa et al. (Sousa et al., 2020) successfully extracted collagen from cod skin under conditions of 37 °C and 50 bar, achieving a yield of 13.8 % after 3 h. The extracted collagen retained its

structure and demonstrated potential for biomedical and cosmetic applications. However, the study did not investigate different experimental conditions. Pho et al. (Phon et al., 2023) used response surface methodology (RSM) and found that under the parameters of 75 bar, 37 °C, and 24 h, the extraction yield of collagen/gelatin was the highest, with a significantly higher yield from fish skin (36.85 %) compared to fish bone (8.1 %). At different pressure levels, higher pressure increased CO₂ density, enhancing its solubility in water and collagen, thereby improving the extraction yield. An appropriate temperature facilitated CO₂ diffusion and dissolution, while too high temperatures (>37 °C) could disrupt the triple-helix structure of collagen. In contrast, Silva et al. (Silva et al., 2016) conducted experiments under different pressure and extraction time conditions and found that increasing them did not significantly improve the yield. The experimental condition of 10 bar for 3 h effectively reduced energy consumption while maintaining optimal extraction efficiency. The difference in experimental results may be due
to the different materials used. Fish skin and fish bones require high pressure to enhance solubility, whereas marine sponges can achieve collagen/gelatin extraction at lower pressure. Sample preparation requires the removal of salts, non-collagen components, and other impurities. However, marine sponges are typically freeze-dried and then ground or milled to approximately 0.5 mm, whereas fish skin is cut into small pieces (10 × 10 mm); the precise grinding steps vary according to the species and the researchers' protocols. Furthermore, not only do different raw materials like fish skin, fish bones, and marine sponges require different pressures for effective collagen extraction, but even among marine sponges, species-specific differences can influence the extraction yield. Barros (Barros et al., 2015) et al. found that the extraction yield of collagen/gelatin using CO₂-acidified water varied among different marine sponge species.

Another issue with fish gelatin is the unpleasant fishy odor (Cui & Li, 2023). The fishy odor is caused by fish oil and lipid oxidation products such as trimethylamine, trimethylamine oxide, and volatile fatty acids as well as non-polar small molecule metabolites (such as amines, aldehydes, and sulfur compounds) (Liu et al., 2024). This is usually mitigated through defatting treatments, activated carbon adsorption, enzymatic degradation, or microencapsulation.

## 2.2. Mathematical modeling of ScCO₂ process

Mathematical models provide us with a method to evaluate system behaviour under various conditions using limited experimental data. ScCO₂ process is highly dependent on the physical states of CO₂, and the parameters governing the physical states as well as interactions and solubility of extracts with the CO₂. To reduce time and cost, mathematical models of ScCO₂ can be utilized or developed. These models enable the optimization of experimental conditions and can be extended to other new system for predicting. There have been many attempts to model and simulate the extraction process to better understand such processes. There are two main models: Thermodynamic model and Kinetic model that are used and the section below discusses the investigations utilising these models. Thermodynamic models explain the maximum amount of extract that can dissolve at equilibrium, whereas kinetic models address how long it takes to complete the extraction. For fish oil extraction from fish waste, the process is generally controlled by thermodynamics in the initial stage and by kinetics in the later stage. In calculating the extraction rate, kinetic models require the solubility under the given conditions, which is determined either through thermodynamic modelling or experimental measurement. The related models are numerous, and this review only selects the commonly used models for discussion (Table 3).

### 2.2.1. Thermodynamic model

Thermodynamic models are primarily used to explore the equilibrium state of a system, the solubility of solutes in ScCO₂, and phase equilibrium relationships, as well as to analyze the effects of temperature and pressure on solubility. Under specific temperature and pressure conditions, the equilibrium solubility determines the maximum yield that can be achieved. Crossover point refers to the temperature at which the impact of temperature on solubility changes from negative to positive under the dual influence of CO₂ density and solute vapor pressure, which is of great significance for optimizing operating conditions. Thermodynamic models can reflect or predict cross over points.

Due to their simplicity, semi-empirical models have broader applicability. Among them, the Chrustil model is the most widely used. The original correlation proposed by Chrustil is presented in Eq. (1).

$$y = \rho^c \exp \left( \frac{A}{T} + B \right) \quad (1)$$

In this equation, y represents the solubility (g/L), ρ denotes the supercritical fluid (SCF) density (g/L), T is the temperature in Kelvin, and A, B, and C are empirical coefficients determined by fitting the data. It describes the linear relationship between the natural logarithm of solute solubility and the logarithm of ScCO₂ density. This equation has been applied to investigate the extraction processes of fish oil (Lopes et al., 2012), carotenoids (Youn et al., 2007), and other substances. However, in the presence of a co-solvent, the semi-empirical model shows a relatively high average absolute relative deviation due to the lack of consideration for co-solvent effects (Zhan et al., 2017). Some authors have adjusted and added model parameters to incorporate the effects of co-solvents, making the models more applicable to ternary systems (González et al., 2001; Mendez-Santiago & Teja, 2000). Lopes et al. (Lopes, Sánchez-Camargo, Ferreira et al., 2012) investigated the solubility of fish oil and the selectivity for EPA and DHA, analyzing solubility behavior and fractionation performance under different temperatures (33–60 °C) and pressures (100–400 bar). The authors used the Chrustil model to correlate solubility with CO₂ density and temperature, verified its applicability for predicting solubility, and calculated the selectivity of CO₂ under different conditions using separation factors. They concluded that Chrustil modeling is suitable for predicting solubility. The Chrustil model, characterized by its simple form and high fitting accuracy, can effectively predict the solubility trends of lipophilic compounds within a certain range. It holds practical value for process optimization and industrial scale-up in ScCO₂ extraction.

Compared to simple semi-empirical models, EoS models have the advantage of being applicable to different types of solutes and solvents as well as complex multi-component co-solvent systems. EoS models are important tools for describing the thermodynamic properties of fluids

<table><caption>**Table 3**
Summary of thermodynamics and kinetic models applied to ScCO₂.</caption><thead><tr><th>Model domain</th><th>Modelling approach</th><th>Characteristics</th><th>Advantages</th><th>Disadvantages</th><th>Reference</th></tr></thead><tbody><tr><td>Thermodynamic</td><td>Chrustil model</td><td>Solute-solvent association equilibrium, density and temperature dependent</td><td>Simple; widely used for many solutes</td><td>Assumes constant k, accuracy decrease at high temperature</td><td>(Chrustil, 1982)</td></tr><tr><td>Thermodynamic</td><td>Peng-Robinson (PR) EoS model</td><td>Cubic EoS based on Van der Waals theory, improve liquid-phase prediction</td><td>Suitable for vapor-liquid equilibrium; applicable to non-polar and slightly polar compounds</td><td>Less accurate for strongly polar or associating compounds; may require mixing rules or volume correction</td><td>(Peng & Robinson, 1976)</td></tr><tr><td>Thermodynamic</td><td>Soave-Redlich-Kwong (SRK) model</td><td>based on Van der Waals theory, introduce α(T)</td><td>Simple; widely used for industry</td><td>Poor accuracy near critical point</td><td>(Soave, 1972)</td></tr><tr><td>Kinetic</td><td>Gompertz model</td><td>Sigmoidal mass transfer behavior</td><td>Simple, fit S-shaped data well</td><td>Poor extrapolation</td><td>(Gompertz, 1825)</td></tr><tr><td>Kinetic</td><td>Goto model</td><td>Two-step mass transfer mode</td><td>Incorporates internal and external mass transfer; easy to fit preliminary extraction data of different materials</td><td>No spatial resolution of internal structure</td><td>(Goto et al., 1993)</td></tr><tr><td>Kinetic</td><td>Sovová model</td><td>Multistage convection-diffusion model</td><td>Physically realistic and interpretable; widely applicable with good accuracy</td><td>Multiple fitting parameters</td><td>(Sovová, 1994)</td></tr></tbody></table>
and are widely used for solubility prediction in SCF systems. Among them, the Peng-Robinson (PR-EoS) model and the Soave-Redlich-Kwong (SRK-EoS) model are the most commonly used ones. The fundamental equation for the PR-EoS is shown in Eq. (2).

$$P = \frac{RT}{v-b} - \frac{a\alpha(T)}{v(v+b) + b(v-b)} \quad (2)$$

In this equation, P represents the system pressure, R is the universal gas constant, T denotes the absolute temperature, v is the molar volume (the volume per mole of substance), b is the co-volume parameter accounting for the finite size of the molecules, and a(T) is the temperature-dependent attraction parameter that characterizes the intermolecular forces. The PR-EoS improves upon the classical Van der Waals equation by incorporating a temperature-dependent parameter, a(T), in the attractive term. This modification allows for a more accurate prediction of phase equilibrium for nonpolar and weakly polar molecules by better reflecting how intermolecular attractions change with temperature. Melgosa et al. (Melgosa Gómez, Sanz Díez, García Solaesa et al., 2017) treated the mixed fish oil as a single pseudo-component, and measured and modelled the phase behaviour of the fish oil-ethanol-SCCO₂ system over the range 10–30 MPa and 323–343 K. The PR equation of state combined with the van der Waals quadratic mixing rule (vdW2) mixing rule was used, in which the binary interaction parameter kij corrects the geometric mean of the attractive term a and lij corrects the arithmetic mean of the repulsive term b to calculate the mixture parameters. The authors compared the fitting performance of PR + vdW2 and PR + Wong-Sandler, concluded that the difference in accuracy was small, and therefore chose the simpler PR + vdW2 approach. This equation demonstrated good applicability in modelling the phase behavior of ternary systems and can serve as an effective simulation tool to guide processes such as ScCO₂ extraction.

Another common equation of state is the SRK-EoS, with its basic equation shown in Eq. (3).

$$P = \frac{RT}{v-b} - \frac{a\alpha(T)}{v(v+b)} \quad (3)$$

In the SRK equation, symbols such as P, R, T, v, b, and a(T) carry the same fundamental meanings as in the PR-EoS. However, while their physical interpretations are identical, the SRK model employs a different formulation for the temperature correction factor a(T) in the attractive term compared to the PR-EoS. SRK-EoS, based on the Redlich-Kwong equation, incorporates an acentric factor to modify the attractive term, enhancing its accuracy in vapor-phase equilibrium calculations. Sovová et al. (Helena Sovová, Stateva & Galushko, 2001) investigated the applicability of the SRK-EoS in predicting the solubility of β-carotene in ScCO₂, with ethanol and vegetable oil as co-solvents. They found that ethanol significantly enhanced the solubility of β-carotene in ScCO₂, with the increase in solubility being proportional to the square root of ethanol concentration. They concluded that the SRK-EoS can effectively describe the solubility behavior of β-carotene in ScCO₂. The research team then extended their study to the overall process of carotenoid extraction from microalgae, proposing that carotenoids are "extracted along with the oil in which they are dissolved." They hypothesized that the solubility of carotenoids depends on the solubility of triolein. Therefore, triolein was introduced into the model, and the EoS was employed to accurately calculate the interactions between triolein and CO₂, thereby providing a more precise simulation of the liquid-vapor phase equilibrium in the ternary system of oil, carotenoids, and CO₂, and revealing the true extraction mechanism of carotenoids (Helena Sovová & Stateva, 2019). It is worth noting that the importance of EoS is when calculating the solubility of food-related solutes. Properly adjusting Tc and Pc can optimize the predictive performance of EoS. In contrast, semi-empirical models do not rely on critical parameters, making them more stable in correlating the solubility of food-related solutes.

### 2.2.2. ScCO₂ extraction process kinetic model

The Gompertz model describes the three phases of ScCO₂ extraction: lag phase, extraction phase, and penetration phase. The basic equation of the model is shown in Eq. (4).

$$y(t) = a \cdot \exp[-\exp(b - ct)] \quad (4)$$

In this equation, y(t) represents the cumulative extraction yield at a certain time. The parameter a denotes the maximum achievable extraction yield, b is a constant associated with the initial lag phase of the extraction process, and c is the extraction rate constant that governs the steepness of the curve. It uses a sigmoidal curve to depict changes in the extraction rate, making it particularly suitable for explaining the lag phase at the beginning of extraction and the drainage stage after reaching the maximum oil yield (Wang & Guo, 2024). The Arrhenius equation describes the effect of temperature on the rate constant and can be applied to modify the equation by introducing temperature dependence into its rate parameter, thereby improving its predictive accuracy (Laidler, 1984). For example, Shalfoh et al. (Shalfoh et al., 2024) adopted the Gompertz equation and Arrhenius law and modified them. By refining the formulas, they improved the dynamic characterization of time dependency. Additionally, they combined experimental data to quantify the effect of temperature on extraction rates, estimate activation energy, and reveal that extraction is more dependent on pressure than temperature.

Given the limitations of empirical models in elucidating extraction mechanisms, the Goto model, as a semi-empirical model incorporating certain mass transfer theory, provides a relatively better mechanistic interpretation of the extraction process. The equation of the Goto model is shown in Eq. (5).

$$m_{\text{ext}}(h=H, t) = \frac{A[\beta/k + (1-\beta)]X_{0p_s}Q_{\text{CO}_2}}{\rho} \left\{ \frac{\tau}{a_1} \left[ \exp\left(\frac{a_1t}{\tau}\right) - 1 \right] + \frac{\tau}{a_2} \left[ 1 - \exp\left(-\frac{a_2t}{\tau}\right) \right] \right\} \quad (5)$$

Where β represents the particle porosity, k is the desorption equilibrium constant, H denotes the bed height, and t is the extraction time. τ refers to the residence time of the solvent in the extraction bed, while X₀ is the initial solute mass fraction in the solid phase, and ρs is the density of the solid material. The terms A, a₁, and a₂ are model parameters derived from specific expressions that characterize the mass transfer behavior. The Goto model describes the desorption-dissolution-diffusion mechanism of solutes within the solid matrix during ScCO₂ extraction, making it more suitable for complex multi-stage extraction processes. Sara Ahmadkelayeh et al. (Ahmadkelayeh et al., 2022) modified this model by linearizing the local equilibrium relationship to account for strong solute-solid interactions during extraction and introducing reaction kinetic constants. Ahmadkelayeh et al. (Ahmadkelayeh et al., 2022) optimized the extraction of lipids and astaxanthin (ASX) from *Pandalus borealis* and carried out kinetic modelling. Using the Goto model to fit the cumulative extraction curves of lipids/ASX, they adjusted only the adsorption equilibrium constant K—determined by minimizing the Average Absolute Relative Deviation percentage (AARD %)—while all other parameters were obtained from experiments and physicochemical/mass-transfer correlations. The authors concluded that the Goto model was able to fit the cumulative extraction curves of lipids and ASX well. The Goto model inherently accounts for K and both internal and external mass transfer resistances, which is essential for systems with strong adsorption and low solubility. However, the authors used a constant K to represent the strength of the solid-solute interaction, whereas in practice K may not be constant and could vary with time, temperature, pressure, and other factors.

Compared to the relatively simplified structure of the Goto model, the Sovová model establishes a more systematic mechanistic framework by distinguishing between different mass transfer stages and resistances. The Sovová model (H. Sovová et al., 1994) is a classical kinetic model
that divides the extraction process into three stages: a fast extraction stage, an intermediate transition stage, and a slow extraction stage, which can be described by Eqs. (6), (7), and (8) respectively, where the extraction curve is defined as a function of time, y(t).

$$y(t) = Q_{CO_2} \cdot y_r \cdot [1 - \exp(-Z)] \cdot t \quad (6)$$

$$y(t) = Q_{CO_2} \cdot y_r \cdot [t - t_{CER} \cdot \exp(z_w - Z)] \quad (7)$$

$$y(t) = m_{SI} \left[ x_0 - \frac{y_r}{W} \ln \left\{ 1 + \left[ \exp\left(\frac{Wx_0}{y_r}\right) - 1 \right] \cdot \exp\left[ \frac{WQ_{CO_2}}{m_{SI}}(t_{CER} - t) \right] \frac{x_k}{x_0} \right\} \right] \quad (8)$$

$Q_{CO_2}$ denotes the flow rate of ScCO₂, $y_r$ is the maximum solubility of the solute in CO₂, and Z is the mass transfer parameter in the solvent phase. $t_{CER}$ refers to the end time of the fast extraction stage, and $z_w$ is a transition parameter related to the characteristics of the extraction bed. In the slow diffusion-controlled stage, $m_{SI}$ is the slope factor of the model, $x_0$ represents the initial solute concentration in the solid phase, $x_k$ is the residual solute amount that remains unextracted, and W is the solid-phase mass transfer parameter. González et al. (González et al., 2001) modified the model to make it more suitable for systems containing a co-solvent. Ioulia Georgiopoulou et al. (Georgiopoulou et al., 2022) directly applied this model and found it to have high fitting accuracy and low prediction error, making it suitable for the extraction of bioactive compounds from Chlorella vulgaris. Similarly, Mouahid et al. (Mouahid et al., 2020) also applied the model; however, they made the hypothesis that the transition stage had little impact on the overall extraction and simplified it into two stages for dynamic analysis. The authors concluded that the simplified model fits the experimental data well, with the average absolute relative deviation as low as 1.33 % (Arrhenius, 1889). Bucio et al. (Bucio, Sanz, Beltrán et al., 2016) extracted fish oil from fish meal and analyzed the extraction kinetics at different temperatures (25–80 °C) and pressures (10–40 MPa). The authors fitted the extraction curves using the Sovová model, dividing them into an initial “solubility-controlled” linear stage and a later “internal-diffusion-controlled” stage. They concluded that the Sovová model’s calculated curves agreed well with the experimental data. The fat in fish meal may be more readily removed by the solvent, with the initial stage mainly limited by solubility and the later stage governed by internal diffusion. By combining thermodynamic and kinetic models, both the solubility and mass-transfer processes can be explained, providing guidance for optimizing pressure and temperature selection.

These models not only reveal the fundamental principles of mass and heat transfer but also provide a scientific basis for optimizing process conditions. However, current research is largely focused on laboratory-scale studies, and the applicability and accuracy of the models in

industrial-scale applications require further validation.

### 3. ScCO₂ fractionation

ScCO₂ fractionation is a process that separates different components in a mixture by leveraging variations in solubility under different temperature and pressure conditions and could be used to separate the extracts for better purity. By adjusting these parameters or collecting fractions at different times, specific components can be enriched. As shown in Fig. 6, different components can be collected in different separators.

In a supercritical fractionation column (as shown in Fig. 7), a controlled temperature and pressure gradient allows selective separation of compounds based on their solubility differences in ScCO₂. In multicomponent systems, the solubility data of individual compounds are crucial for selective fractionation, but the co-solute effect may influence the solubility of the target compound (Kfoury & Fourmentin, 2024). The goal of fractionation is to separate different components in a mixture by controlling the solubility and flow properties of ScCO₂. Because the solubility of the target compound varies under different temperature and pressure conditions, fractionation can be achieved by operating at different temperatures and pressures, or collecting at different times, to obtain fractions rich in specific components. Fatty

<figure><img src="image_13.png" /><figcaption>Fig. 7. Fractionation tower.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>Fig. 6. Schematic Diagram of ScCO₂ Extraction and Fractionation System.</figcaption></figure>
acid ethyl ester (FAEE), with a simpler structure than triacylglycerol (TAG), dissolves more easily in ScCO₂, facilitating omega-3 fractionation. FAEE is typically obtained via transesterification of extracted fish oil (Melgosa et al., 2021). In the fractionation column, ScCO₂ enters from the bottom and flows upward, while the liquid FAEE mixture flows downward from the top. The short-chain FAEE is dissolved by ScCO₂ and exits from the top of the column, while the remaining liquid at the bottom is enriched with long-chain FAEE. This countercurrent fractionation can effectively enrich the target component. Increasing the reflux ratio will improve the purity of the light phase but reduce the overall yield, requiring a trade-off for optimization (Fernandes et al., 2011). Current research has successfully achieved the enrichment of omega-3 (Létisse & Comeau, 2008), squalene (Al-Darmaki et al., 2012), phospholipid (Taylor & King, 1998), and other compounds using ScCO₂. In omega-3 fractionation, experimental parameters can be adjusted to control the EPA/DHA ratio. Generally, within a certain range, higher CO₂ flow rate and pressure lower the EPA/DHA ratio (Perretti et al., 2007). Conducting experimental validation in a fractionation column is necessary (Riha & Brunner, 2000), but the actual investment cost for the fractionation column is relatively high. For a plant with a feed rate of 10 kg/h, the equipment investment cost is about 1.6 million euros; for a 100 kg/h plant, it is around 3.9 million euros; and for a 300 kg/h plant, it is approximately 6.3 million euros (Fiori et al., 2014). Thus, simplifying experimental equipment and developing simulation models (Espinosa et al., 2002; Fiori et al., 2014; Schembri et al., 2023) provides new approaches for research.

Unlike column fractionation, which typically processes refined oils, the fractionation of raw materials requires tuning multiple experimental parameters. By optimizing temperature, pressure, co-solvent composition, and separator conditions in the ScCO₂ system, and considering solubility characteristics, target compounds can be effectively enriched. Rodrigues et al. (Rodrigues et al., 2021) set up two collection vessels with different temperatures and pressures, enabling fractionation of extracts collected over 135 min. The first vessel, operating at 35 °C and 60 bar, primarily extracted non-polar and moderately polar compounds, such as neutral lipids and monounsaturated fatty acids (MUFA), during the early phase (0–30 min). The second vessel, under higher temperature and near atmospheric pressure, gradually extracted more polar compounds, including polyunsaturated fatty acids (PUFA) and phospholipids, during the mid-to-late phase (30–135 min). During the extraction process, the polarity of the extract gradually increases with the gradual addition of the co-solvent (Bastante et al., 2022). Future research should focus on refining operational parameters, exploring co-solvent effects, and enhancing process efficiency to further optimize ScCO₂-based separation techniques for industrial applications.

## 4. Extraction enhancement with other complementary techniques

While ScCO₂ extraction has many advantages, the extraction efficiency can be enhanced by combining the process with other complementary or pretreatment procedures such as ultrasound (Ou et al., 2024) and microwave (Yousefi et al., 2024). Ultrasound facilitates the disruption of cell walls through cavitation effects caused by high-frequency sound waves, thereby increasing the contact area between the solvent and the cell's internal components and accelerating mass transfer. Fa et al. (Fan et al., 2015) demonstrated through a kinetic model that the extraction rate of ultrasound-enhanced subcritical CO₂ extraction (k = 0.6599) was higher than that of conventional ScCO₂ extraction (k = 0.5683), proving that ultrasound accelerated the release and dissolution of lutein, thereby improving extraction efficiency. Mai et al. (Mai et al., 2023) found that under optimized ultrasound conditions (60 °C, 30 min, with 1 g of NaCl), the extraction efficiency increased from 47.9 % to 54.7 %. Nunes et al. (Nunes et al., 2021) combined microwave (MW) pretreatment with ScCO₂, where MW radiation effectively disrupted the rigid exoskeleton of crab shells,

enabling efficient recovery of astaxanthin (ASX) from marine crustacean waste, particularly from crabs. MW pretreatment enhanced ASX recovery and reduced extraction time by 90 % compared to traditional methods.

In addition, ScCO₂ mainly extracts lipophilic compounds, while subcritical water (sw), enzymatic hydrolysis, ultrasound-assisted, microwave-assisted and pulsed electric fields (PEF) techniques can extract polar and water-soluble components such as antioxidants, polysaccharides, and protein hydrolysates. By combining ScCO₂ with these different extraction techniques, Gouveia et al. (Gouveia et al., 2021) found that the sw extraction combination was the most effective in the ScCO₂ extraction of *Tetradesmus obliquus*, yielding the highest extraction rate and antioxidant content. J. López-Limón et al. (López-Limón et al., 2025) used ScCO₂ pretreatment to disrupt the cell structure of *Spirulina (Arthrospira maxima)* to obtain C-phycocyanin. Melgosa et al. (Melgosa et al., 2020) combined ScCO₂ and sw technologies to extract various products: ScCO₂ technology was used for the efficient extraction of fish oil, enhancing protein extraction purity through defatting. Sw extracted protein hydrolysates via hydrolysis, making it suitable for producing high-value functional components. Badfar et al. (Badfar, Jafarpour, Casanova et al., 2025) applied ScCO₂ defatting to shrimp by-products prior to enzymatic hydrolysis using Alcalase and Trypsin, resulting in the production of functional peptides with antioxidant activity. Liu et al. (Liu, Berrada, Castagnini et al., 2025) employed ScCO₂ defatting followed by PEF-assisted extraction to efficiently recover proteins, bioactive peptides, minerals, and antioxidant components from salmon side streams. The integration of these synergistic technologies not only improves extraction efficiency but also reduces extraction time and energy consumption while increasing the variety of extracted compounds. This multi-technology combination strategy holds great potential in the development of biological resources and the extraction of high-value-added products (Table 4).

<table><caption>**Table 4**
Summary of extraction enhancements using complementary techniques.</caption><thead><tr><td>Technique</td><td>Mechanism</td><td>Benefits</td><td>Limitations</td><td>Reference</td></tr></thead><tbody><tr><td>Ultrasound</td><td>Cavitation breaks cell walls</td><td>Higher yield, reduced extraction time</td><td>Limited for heat-sensitive compounds</td><td>(Ou et al., 2024)</td></tr><tr><td>Microwave</td><td>Internal heating, cell rupture</td><td>Higher yield, reduced extraction time</td><td>Limited for heat-sensitive compounds</td><td>(Nunes et al., 2021)</td></tr><tr><td>Enzyme</td><td>Peptide bond cleavage</td><td>Mild conditions, high specificity</td><td>Enzyme residue contamination; high cost</td><td>(Badfar, Jafarpour, Casanova et al., 2025)</td></tr><tr><td>Subcritical water (sw)</td><td>Lowering water's dielectric constant and increasing ionization at high temperature and pressure to enhance solubility and hydrolysis</td><td>Extract polar and water-soluble components</td><td>High temperature and pressure</td><td>(Gouveia et al., 2021)</td></tr><tr><td>Pulsed electric fields (PEF)</td><td>Electroporation, cell lysis</td><td>Non-thermal, higher yield, reduced extraction time</td><td>High cost</td><td>(Liu, Berrada, Castagnini et al., 2025)</td></tr></tbody></table>
## 5. Economic feasibility

Several economic feasibility studies have indicated the potential of ScCO₂ for large-scale commercial production. Fiori et al. (Fiori et al., 2014) investigated the ScCO₂ fractionation process for fish oil-derived omega-3 FAEES, designing three SFF plants with feed capacities of 10, 100, and 300 kg/h. The corresponding investment costs were reported as €1.6 million, €3.9 million, and €6.3 million, respectively. They further went on to project the unit product costs by assuming continuous operation for 300 days per year, 24 h a day and taking into account both production output and capital recovery, the unit product costs were calculated as €17.43/kg (10 kg/h), €5.97/kg (100 kg/h), and €4.27/kg (300 kg/h). The study concluded that economic feasibility is highly dependent on processing scale. Due to the high initial capital investment, small-scale systems may result in prohibitively high unit costs and are not commercially attractive, whereas medium- and large-scale operations demonstrate good economic viability. Another study by Shalfoh et al. (Shalfoh, Ahmad, Binhweel et al., 2024) conducted an economic feasibility analysis on the extraction of fish oil from fish waste for biodiesel production. They found that in actual production, feedstock cost accounts for approximately 75–80 % of the total cost, making it the main cost factor. Using waste as feed stock is critical in reducing costs and consequently affects the feasibility of the process. The study concluded that using ScCO₂ technology to extract oil from fish waste for biodiesel production is economically feasible, particularly in countries with abundant fish waste resources and low feedstock acquisition costs. Zgheib et al. (Zgheib, Saade, Khallouf et al., 2018) conducted an economic assessment of astaxanthin extraction from microalgae. The costs included raw materials, energy, labor, and management. They found that the breakeven point was a market price of $1500/kg for natural astaxanthin. If the market price reaches $6000/kg, the ROI can reach 113 %, with a payback period of one year. Therefore, ScCO₂ extraction is economically feasible, especially in the field of high-value-added products.

In summary, to ensure sustainable large-scale production using supercritical fluid (SCF), it is essential to conduct process-based modelling studies, optimize the process, control raw material costs, and aim to produce high-value products whenever possible. Overall, industrial-scale production using SCF demonstrates a certain degree of economic feasibility and aligns well with the current trend of green economy development.

## 6. Challenges and limitations

The high initial investment cost of pressurized equipment may pose a challenge. This necessitates a thorough evaluation of economic feasibility, especially for industrial implementation. A general guideline is to make the process economics more viable by targeting high-value products (Vardanega et al., 2022), controlling the cost of raw materials (Shalfoh, Ahmad, Binhweel et al., 2024), and reducing solvent loss (Pinto et al., 2022).

To standardize and regulate various ScCO₂ systems for different applications, a more unified approach is needed. Currently, while ScCO₂ systems used for dissolving different solutes have similar compositions, their reaction vessels vary due to different solubility requirements. Establishing more standardized ScCO₂ systems would allow for flexible functional switching by simply replacing reaction vessels to accommodate different solutes.

## 7. Conclusion and future directions

To conclude, ScCO₂ provides us with a greener and sustainable method for obtaining valuable compounds with several aspects requiring further improvements. Firstly, process optimization is crucial. The extraction and fractionation parameters vary depending on the equipment used and the characteristics of the raw materials. Although

many studies work on parameter optimization through Box Behnken design and others, a universally applicable screening method has yet to be established. Beyond optimizing the condition parameters, the sample preparation process should be optimized to get high-quality and high-yield products. Secondly, attention should be paid not only to extraction yield but also to purity. Thirdly, many raw materials contain multiple valuable compounds, yet current research concentrates on the extraction of single compounds. The residue of materials after extraction may have other valuable compounds. Therefore, it is important to analyze the composition and transformation of these remaining compounds after ScCO₂ extraction. Future research should explore strategies for single-run or multi-run extraction of multiple components to enhance resource utilization. Fourthly, can explore special additives or modifiers to improve solubility and diffusion dynamics of target compounds in ScCO₂. Fifthly, currently many studies lack a systematic evaluation of economic factors such as cost, return on investment, and energy consumption, which are vital for scaling up and industrial application. Finally, supercritical conditions are unsafe due to high pressure. Thus, there is a pressing need to establish a real-time and precise online monitoring and control system to ensure the product consistency and experimental safety.

## Ethical statement

N/A: The research presented does not involve any animal or human study

## Claim

During the preparation of this work the authors used “DeepL Write” to double-check for English spelling or grammatical errors or typos. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

## CRediT authorship contribution statement

**Qiqi Sun:** Writing – original draft, Visualization, Conceptualization, Funding acquisition. **Jean-Christophe P. Gabriel:** Writing – review & editing, Supervision, Conceptualization, Funding acquisition. **Lay Poh Tan:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jean-Christophe P. Gabriel reports financial support was provided by the Agence Nationale de la Recherche, the National Environment Agency as well as from the National Research Foundation, Singapore. Qiqi Sun and Tan Lay Poh reports financial support was provided by Singapore Food Agency, NTUitive and MOE (Singapore).

## Acknowledgements

QS & TPL acknowledge financial support from Singapore Food Agency (Seed grant), NTUitive and MOE (Singapore). JCG acknowledge financial support: (i) for the SCARCE facilities from National Research Foundation, Singapore, and National Environment Agency, Singapore under its Closing the Waste Loop Funding Initiative (Award CTRL-2023-1D-01); (ii) from the French government managed by the Agence Nationale de la Recherche under the France 2030 programme, under the reference ‘ANR-22-PERE-0003’.

## Data availability

Data will be made available on request.
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