Preparation of biocrude based on fish proteins and lipids as feedstock by subcritical hydrothermal liquefaction

As human demand and fishing technologies have developed, global marine fisheries have gradually expanded. According to a 2023 report, the global production of aquatic products reached ∼186.6 × 10⁶ tons, with around 35% considered waste and discarded—an enormous figure.^1,2 The disposal of this seafood waste presents a significant challenge. Many countries worldwide have introduced policies for handling seafood waste. For example, the United Nations Sustainable Development Goals (SDGs) emphasize the need to reduce seafood waste and enhance the use of seafood by-products.³ Iceland’s “100% Fish Utilization” initiative highlights the value of fish by-products through technological innovation.⁴ Namibia, a relatively underdeveloped nation economically, has announced plans to use fish skin for collagen extraction in pharmaceuticals and cosmetics, providing local communities with more economic opportunities and reducing environmental pressure.³ Clearly, the world faces substantial challenges related to seafood waste disposal and environmental pollution.

According to previous research, there are various methods for handling seafood waste. For example, Kannan et al. used hydrothermal carbonization to produce biochar from fish waste, with a biochar yield of around 35%.⁵ Although this technique is environmentally friendly, it consumes a significant amount of energy in the production process and has a relatively low yield. Prakash et al. investigated extracting chitin and chitosan from waste for use in biomedicine and agriculture. This method is low-cost and pollution-free, but after extracting the desired substances, a large amount of fish meat and bones remain as waste, with very low utilization rates.⁶ Essabiri et al. suggested using the waste as biofertilizer, converting discarded fish into organic fertilizer or liquid fermentation feed through composting.^6,7 However, seafood waste may contain pathogens or heavy metals, and inadequately treated waste during composting could result in pathogen residues or heavy metal buildup, affecting the safety of the compost product. Additionally, the leachate produced during composting may contain large amounts of nitrogen, phosphorus, and other elements, which, if improperly managed, could pollute soil and water sources.⁷ Venugopal et al. explored the concept of biorefinery, using micro-organisms or enzymes to convert seafood waste into nutrients, proteins, and oils for human and animal food.⁸ Although biorefining can extract high-value components, the by-products (such as solid residues and nitrogen-rich wastewater) still require further treatment; otherwise, they may pollute the environment. Clearly, there is an urgent need to develop a technology for processing seafood waste that is both environmentally friendly and capable of achieving high yields and high-value outcomes.

HTL is an emerging thermochemical technology that converts organic materials into liquid fuels under high temperature and pressure conditions. It is typically applied to biomass conversion. HTL simulates the chemical reaction processes in the high-temperature and high-pressure environments found underground in nature, enabling the conversion of water-rich biomass into oil, gas, and solid products. This technology achieves a material utilization rate of 70%–90%,⁹ with gas, solid, and liquid products all being useable resources. Furthermore, it is an environmentally friendly technology that does not produce harmful gases, has low particulate emissions, and, compared to traditional thermal conversion technologies (such as gasification and carbonization), can process a variety of feedstocks while avoiding energy-intensive drying processes,¹⁰ thereby reducing unnecessary energy consumption.^11,12

At present, most of the research on biomass HTL focuses on green algae or terrestrial plants.^13–15 It is because these substances are widely sourced and have high product yields. However, these biomass raw materials do not have as intense an impact on the environment as fishery waste does. Fishery waste is one kind of biomass, and most of the fishery waste is fish. Its main components are protein and carbohydrates, and these components are substances that are easy to decompose at high temperatures.^16,17 While plants contain a large amount of cellulose, which is a substance that is difficult to decompose and makes the HTL process require more energy consumption. The temperature needed for their HTL is mostly between 291 and 395 °C,¹⁸ while for fish biomass with a relatively high protein content, the HTL temperature is usually between 200 and 300 °C.¹⁷ Undoubtedly, treating fishery waste through HTL can relieve environmental pressure and also provide a continuous source for fossil fuels. This experiment was carried out in Zhoushan City, Zhejiang Province, China. This area has a high yield of small yellow croakers, and they are easy to obtain. Therefore, in this study, small yellow croakers are taken as the research object instead of fishery waste. Besides, this article aims to study the mechanism of HTL of fishery waste and find the most suitable reaction conditions and the changing trend of product components at different temperatures.

Small yellow croaker was randomly selected from various seafood stores in Zhoushan, Zhejiang Province. The fish were dried in an oven at 60 °C for 30 min and then crushed for ∼3 min using a universal high-speed crusher.

The critical point of water is at a temperature of 374.2 °C and a pressure of 22 100 kPa. When the temperature and pressure of water exceed these critical conditions, it is referred to as supercritical water. Under certain pressures, heating liquid water into a gaseous state surpasses the liquid–gas boundary line. Water below this critical point is termed subcritical water. Supercritical and subcritical water possess unique properties that are different from those of ordinary water, such as a lower dielectric constant, reduced hydrogen bonding, lower viscosity, higher solubility, and catalytic activity. These properties make supercritical and subcritical water better for diffusion with lower surface tension, making it easier to dissolve organic matter.

Table I lists some properties of ordinary, subcritical, and supercritical water, highlighting the uniqueness of subcritical water. From Table I, it is evident that subcritical water has a lower dielectric constant of 14.0 Fm⁻¹, which increases the solubility of free fatty acids.¹⁹ Additionally, the ion product constant K_w of water increases under subcritical conditions, indicating the production of more H⁺ and OH⁻ ions. This implies that more acids or bases participate in catalytic reactions, accelerating the hydrolysis of biomass.²⁰ Furthermore, compared to supercritical water, subcritical water has a higher density and ion product constant, favoring ionic reactions. Therefore, this experiment was conducted under subcritical conditions.

The total acid value of biocrude was determined according to the ASTM D664 standard of the American Society for Testing and Materials (ASTM).²⁶ The viscosity of the oil was measured using a DSY-104 kinematic viscometer.²⁷ 

The oil sample was analyzed using an Agilent 7890A-5975C GC-MS system (Agilent Technologies, USA). The chromatographic column used was DB-WAX (30.0 m × 250 µm, 0.25 µm). The initial temperature was set at 40 °C and held for 5 min, then increased to 120 °C at a rate of 5 °C/min, followed by an increase to 230 °C at a rate of 10 °C/min, held for 15 min, and finally increased to 240 °C at a rate of 15 °C/min and held for 5 min. Helium (He) was used as the carrier gas with a flow rate of 1.0 ml/min. The split ratio was 10:1, and the injection volume was 1 μl. The mass spectrometry conditions were set as follows: electron ionization (EI) source with an electron energy of 70 eV, ion source temperature of 230 °C, quadrupole temperature of 150 °C, and a scan mass range of 35–500 u. The oil samples were filtered through a 0.22 μm microporous membrane before direct injection into the GC-MS for analysis.

From the macroscopic observations of the solution, as shown in Figs. 2 and 3, the oil yield is synergistically related to temperature. At 200 °C, the solution appeared as a dark brown suspension without biocrude production. When the temperature reached 250 °C, the solution started to show significant stratification: the lower layer contained oily water, appearing as clear yellow, whereas the upper layer consisted of viscous biocrude, with an oil yield of 25.4%. From a microscopic perspective, as shown in Fig. 4, the essence of biomass liquefaction is the breakdown of biological cells. Various cellular components (proteins, polysaccharides, lipids, etc.) degrade and transform into multiple compounds.²⁸ After the cell membrane breaks, intracellular fluid and organelles are suspended in the solution, primarily composed of proteins, polysaccharides, lipids, cholesterol, and phosphates.²⁹ These substances undergo high-temperature pyrolysis to form new compounds, resulting in the formation of a suspension, whereas biocrude is not produced at this temperature. This indicates that at 200 °C, the reactions necessary for biocrude formation (cracking, recombination, and rearrangement) have not yet occurred. This also suggests that at 200 °C, more substances detrimental to biocrude quality may be produced. Based on this phenomenon, we divided HTL into P₁ and P₂. Where the R point is the temperature at which it transitions to. These results indicate that the R point is between 200 and 250 °C, implying that the temperature for fish biomass HTL must exceed the R point to begin producing biocrude. This provides a reference for temperature settings in subsequent studies, enabling the effective control of reaction conditions and reducing unnecessary by-products.

In this experiment, we conducted a preliminary analysis of the properties of biocrude using three physical indicators: acid value, kinematic viscosity, and density. The physical properties of biocrude can vary depending on the raw materials and reaction pathways used in its production. Therefore, we first identified the physical properties of the produced biocrude. Additionally, based on changes in these properties, we used GC-MS to perform a chemical analysis of the biocrude, focusing on carbon chain distribution and component distribution, to investigate their impact on the physical properties of the biocrude.

From Table II, it can be seen that as the temperature increased, the density of the biocrude gradually decreased. The most significant drop in density occurred between 275 and 300 °C. From Fig. 5, it is evident that at 300 °C, the carbon chains were mainly distributed between C2 and C9, whereas at other temperatures, they were mostly distributed between C16 and C22. Typically, the density of biocrude is directly proportional to the length of its carbon chain. This is likely because longer carbon chains result in an increase in molecular weight and stronger intermolecular forces.³⁰ The distribution of biocrude carbon chains in the lower range helps improve the combustion efficiency. Short-carbon-chain biocrudes produce less carbon deposition during catalytic reforming, extending catalyst life, reducing process costs, and lowering CO₂ emissions, thus providing better environmental benefits.^31,32 Additionally, the viscosity decreases with increasing temperature. Cai et al. proposed that the viscosity of biocrude is highly dependent on temperature, significantly decreasing as the temperature increases, which is a consistent phenomenon across biocrudes from different biomass sources.³³ This aligns well with the results of the present study. Low-viscosity biocrude is more easily degraded by micro-organisms, thereby reducing environmental pollution. Furthermore, research has indicated that low-viscosity biocrude can reduce fuel consumption by ∼2%.³⁴ From the above-mentioned analysis, a reaction temperature of 300 °C and residence time of 125 min can help improve the quality of biocrude.

From Table II and Fig. 6, it can be seen that the acid value decreased with increasing temperature. At 200 °C, the content of fatty acids and their derivatives was as high as 60.1%. Studies have shown that fatty acids and their derivatives readily undergo hydrolysis reactions at high temperatures in the presence of water, producing free fatty acids, which are a significant source of acidic components in biocrudes.³⁵ This led to an increase in the acid value. Additionally, as the temperature increased, the content of fatty acids and their derivatives in the oil significantly decreased. When the temperature reached 300 °C, the content of fatty acids and their derivatives was almost zero, whereas the content of aromatic compounds significantly increased. These aromatic compounds mainly include styrene, ethylbenzene, toluene, and benzene, all of which contain aromatic rings. This is likely due to the deoxygenation reactions of fatty acids to form hydrocarbons.³⁶ Fatty acids at high temperatures may also be converted into epoxides, acetals, ketones, and other compounds, where cyclization reactions can lead to the formation of aromatic compounds.³⁷ At 275 °C, there was a significant increase in amide compounds, such as dodecanamide, N,N-diethyl-, N-methyldodecanamide, 9-octadecenamide, N,N-dimethyl-, and 3-cyclopentylpropionamide, N,N-dimethyl-. The primary source of nitrogen compounds is the decomposition of proteins and amino acids. Under high-temperature conditions, the reactions of nitrogen-containing compounds become more intense, and amides are released during the breakdown of proteins and amino acids, leading to an increase in amide compounds. The release of amide compounds peaked at 275 °C. However, when the temperature increased to 300 °C, the amide compounds almost disappeared, likely due to their decomposition at higher temperatures. It has been reported that amide compounds completely decompose between 280 and 400 °C,^38,39 which is consistent with the results of this experiment. At 300 °C, large amounts of amines are produced, such as 2-propanamine, N-allyl-N,N-dimethylamine, cyclohexanamine, N-butyl-benzenamine, 4-methoxy-, and 2-propen-1-amine, N-ethyl-N-methyl-. These compounds contain amino groups (-NH₂, -NR₂, or -NRR′), likely resulting from the cleavage of nitrogen-carbon bonds in amides, forming amines that further react to generate complex organic amines. Additionally, as shown in Fig. 6, there was a noticeable increase in pyrrole and piperidine compounds at 300 °C, which were absent at temperatures below 300 °C. These nitrogen-containing heterocycles are formed through hydrothermal reactions of proteins and various amino acids, generating numerous polycyclic nitrogen heterocycles that further decompose into pyrrole and piperidine compounds. Overall, increasing the temperature significantly reduced the acid value of the biocrude. It also intensifies cyclization reactions, producing more aromatic compounds and nitrogen-containing heterocycles, such as piperidine and pyrrole. These substances enhance the combustion performance of biocrude and significantly reduce its oxygen content and acidity, thereby improving its stability.⁴⁰ Furthermore, fatty acids and their derivatives are widely used in the industry for manufacturing plastics, lubricants, and fuels, providing a temperature reference for producing fatty acids and their derivatives through fish biomass HTL (Table III).

We also investigated the changes in the biocrude composition based on different reaction times. As shown in Table IV and Fig. 7, the overall composition mainly includes fatty acids and their derivatives, amides, alcohols and their derivatives, amines, aldehydes, sulfides, aromatic compounds, ketones, pyrroles, piperidines, and alkenes. However, the contents of these components vary significantly with different residence times. When the residence times were 95 and 185 min, the composition and content were similar. Likewise, when the residence times were 125 and 155 min, the composition and content were also similar. Although the components are largely similar, the varying amounts of each have a significant impact on the quality of the biocrude. At 95 and 185 min, the content of fatty acids and their derivatives was very high, which can increase the acidity of the biocrude and reduce its overall stability. This may be because at 95 min, the initial reactions of proteins and amino acids produce a large amount of fatty acids and their derivatives, and a significant amount of fatty acid cracking and reactions occur at this stage. When the time is extended to 185 min, secondary cracking may occur, producing a large amount of phenols and alcohols from the previous reactions, which then further crack to produce more fatty acids and their derivatives.⁴¹ Additionally, the biocrude at these two residence times contained a higher amount of alcohols and their derivatives, possibly due to the breakdown of amide compounds into carboxylic acids, which then degraded to form more alcohols. Alcohols generally have a lower heating value than hydrocarbons;⁴² therefore, an increase in alcohols can reduce the overall heating value of the biocrude. Moreover, it can lead to the formation of more harmful by-products during combustion, such as aldehydes and ketones,⁴³ and alcohols are highly corrosive, making the storage and transportation of biocrude problematic. Therefore, residence times of 95 and 185 min are unfavorable for the quality of biocrude.

When the residence times were 125 and 155 min, there were significant changes in alcohols and their derivatives, aromatic compounds, ketones, and alkenes. At 155 min, the content of alcohols and their derivatives was similar to that at 185 min, both being relatively high, and amide compounds were absent. This indicates that between 125 and 155 min, amides decompose significantly, producing a large amount of carboxylic acids, which are further converted into alcohols. As mentioned earlier, an increase in alcohol content lowers the overall quality of biocrude. Additionally, there was a noticeable increase in ketones and alkenes, while aromatic compounds decreased. Studies suggest that an excess of ketones can lead to incomplete combustion of biocrude, resulting in the production of CO gas and some unburned hydrocarbons.⁴⁴ Although alkenes and aromatic compounds significantly improve the fuel performance of biocrude, their content (9.9%) at 155 min is much lower than that in the biocrude obtained with a 125-min residence time (17.22%). Notably, at 125 min, the total content of alcohols and ketones was 3.05%, which was much lower than the 16.45% at 155 min. This indicates that controlling the residence time to 125 min significantly improved the quality of the biocrude.

Overall, a residence time of 125 min resulted in a biocrude composition with more substances beneficial for fuel performance. At residence times of 95 and 185 min, higher amounts of bio-fatty acids and their derivatives were produced. Although these compounds can adversely affect biocrude quality, they can be converted into useable biocrude through the addition of catalysts in subsequent processes or used as references for producing other high-value products. Conversely, a residence time of 155 min leads to the generation of more ketones, alcohols, and alkenes, all of which have a high utility value for fuel additives and medical applications.

In this study, the transition temperature point R from the P₁ to the P₂ was detailed, revealing that R is in the 200–250 °C range. The reactions in the P₁ and P₂ stages were described from both microscopic (cells and compounds) and macroscopic (surface phenomena) perspectives. Additionally, the physical properties of the biocrude were found to be closely related to its composition. Using GC-MS, the effect of temperature changes on the physical properties of the biocrude was analyzed in detail. It was found that while the composition remained similar as the temperature increased from 200 to 300 °C, the content changed significantly. The carbon chain distribution of compounds shifted to lower ranges, resulting in lighter oils, which reduced the viscosity and density of the biocrude. As the temperature increased, fatty acids and their derivatives decreased significantly, whereas aromatic compounds increased, leading to a significant reduction in the acid value and an improvement in the fuel performance of the biocrude. Interestingly, by altering the reaction residence time, it was found that the biocrude compositions at 95 and 185 min were similar, both containing high amounts of fatty acids and their derivatives, resulting in higher acid values. Biocrude compositions at 125 and 155 min were also similar, but the 125-min residence time yielded more substances that improved biocrude quality, particularly aromatic compounds, and had much lower amounts of alcohols and ketones compared to 155 min. Overall, a temperature of 300 °C and residence time of 125 min were highly beneficial for producing high-quality biocrude. Additionally, adjusting the temperature and residence time allows for the control of the content of various compounds to produce related high-value products.

This work was supported by the Bureau of Science and Technology of Zhoushan Project (No. 2021C31002) and Dinghai District of Zhoushan Science and Technology Project (No. 2021C31002).

The authors have no conflicts to disclose.

Junjie Qin: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Methodology (equal); Resources (equal); Software (equal); Writing – original draft (equal). Yan Wang: Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Resources (equal); Supervision (equal). Qinhong Wei: Data curation (equal); Formal analysis (equal); Methodology (equal); Project administration (equal). Yong Chen: Data curation (equal); Funding acquisition (equal); Methodology (equal); Project administration (equal); Resources (equal); Writing – review & editing (equal). Shuqing Yang: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Project administration (equal). Xianmin Zheng: Conceptualization (equal); Funding acquisition (equal); Investigation (equal); Resources (equal); Software (equal).

Data sharing is not applicable to this article as no new data were created or analyzed in this study.