Physical Cell Disruption Technologies for Intracellular Compound Extraction from Microorganisms

Abstract

This review focuses on the physical disruption techniques in extracting intracellular compounds, a critical step that significantly impacts yield and purity. Traditional chemical extraction methods, though long-established, face challenges related to cost and environmental sustainability. In response to these limitations, this paper highlights the growing shift towards physical disruption methods—high-pressure homogenization, ultrasonication, milling, and pulsed electric fields—as promising alternatives. These methods are applicable across various cell types, including bacteria, yeast, and algae. Physical disruption techniques achieve relatively high yields without degrading the bioactivity of the compounds. These techniques, utilizing physical forces to break cell membranes, offer promising extraction efficiency, with reduced environmental impacts, making them attractive options for sustainable and effective intracellular compound extraction. High-pressure homogenization is particularly effective for large-scale extracting of bioactive compounds from cultivated microbial cells. Ultrasonication is well-suited for small to medium-scale applications, especially for extracting heat-sensitive compounds. Milling is advantageous for tough-walled cells, while pulsed electric field offers gentle, non-thermal, and highly selective extraction. This review compares the advantages and limitations of each method, emphasizing its potential for recovering various intracellular compounds. Additionally, it identifies key research challenges that need to be addressed to advance the field of physical extractions.

1. Introduction

Intracellular compounds, ranging from proteins to lipids to pigments, are crucial in various industries, including biotechnology, biofuels, and pharmaceuticals [1,2,3,4]. These compounds are essential for producing bio-based products that serve as key ingredients in animal feed, food, and medical applications. For instance, proteins derived from algal proteins are increasingly used as nutritional supplements or as animal feed ingredients [5]. The extraction of intracellular compounds from microbial cells, particularly those produced through fermentation processes, is a critical step that determines the yield, purity, and functionality of the final products. In fermentation, microorganisms like bacteria, yeast, and algae are cultured to produce a wide array of intracellular compounds. These compounds have diverse applications, from biofuels, where algal lipids are converted into renewable energy sources, to pharmaceuticals, where intracellular proteins contribute to the development of therapeutic agents [6]. Furthermore, pigments synthesized intracellularly through fermentation are used in various sectors, including food and cosmetics, underscoring the broad utility of these bio-derived substances [7]. Efficient recovery of these compounds is, therefore, vital to both the functionality of the final product and the efficacy and sustainability of biomanufacturing processes [6].

Downstream processing, which involves the recovery and purification of intracellular compounds, is a crucial phase to ensure product yield and quality [8]. However, traditional chemical methods for intracellular compound extraction often struggle to achieve both recovery efficiency and the preservation of compound integrity. Solvent extraction, a widely used approach, can degrade sensitive intracellular compounds, compromising their stability and functionality. Moreover, the environmental impact of solvent use—along with the high operational costs associated with solvent recovery, waste management, and disposal—pose significant sustainability and economic challenges [9]. Furthermore, scalability poses an additional barrier, as techniques that work well at a laboratory scale may not be transferable to industrial-scale applications [10,11]. To address some of these limitations, biological approaches such as the enzymatic disruption of cell walls are also employed for intracellular compound extraction. Enzymes offer a more selective and gentler approach to cell disruption, targeting specific cell walls or membrane components to release desired intracellular compounds. One of the key advantages of enzymatic methods is their environmentally friendly nature, requiring lower energy inputs and reducing the need for harsh chemicals. Moreover, they are highly effective at preserving the functionality and structure of intracellular compounds. However, enzymatic processes are hindered by their slow reaction rates and high operation costs. Their efficacy can be inconsistent, varying with different cell types [12], and the need for precise control over reaction conditions, such as temperature and pH, adds to the complexity and cost of the process [13]. Moreover, enzymatic extraction faces challenges in scaling up for industrial applications. The cost and limited availability of certain enzymes, along with the difficulty of replicating optimal process conditions required for enzymes, can further restrict their application in large-scale operations.

Given the limitations of chemical and enzymatic methods, there has been a growing focus on the development of physical disruption techniques for intracellular compound extraction. These methods, which include high-pressure homogenization, ultrasonication, mechanical milling, and pulsed electric field, primarily apply physical forces to disrupt the cell wall or membrane, allowing the release of intracellular products. They offer a promising alternative to conventional chemical and biological approaches [14,15,16,17,18,19]. The shift toward physical methods is driven by their potential to enhance extraction efficiency, reduce operational costs, and minimize environmental impacts [20]. Ultrasonication, for example, uses high-frequency sound waves to induce cavitation phenomena in the liquid medium and within cells. When these bubbles collapse, they generate localized shear forces that can break open microbial cells, making this method particularly effective for disrupting a wide range of cell types [14]. High-pressure homogenization, on the other hand, forces cell suspensions through a narrow orifice at extremely high pressure, causing intense high shear forces that rupture the cells [16]. These methods offer a viable path to cost-effective and environmentally friendly intracellular compound extraction. These methods have also been shown to achieve high yields of target valuable intracellular compounds while preserving their bioactivity. This makes them ideal for industries that rely on the recovery of functional proteins, lipids, pigments, and other high-value products from microbial cells. As industries continue to prioritize green technologies, physical disruption methods present a viable path toward large-scale, sustainable, and cost-effective intracellular product recovery.

In this review, we delve into the principles and applications of various physical cell disruption techniques for the recovery of intracellular compounds. By investigating methods like high-pressure homogenization, ultrasonication, milling, and pulsed electric fields, we aim to highlight how these physical approaches are transforming the field of intracellular compound extraction and being investigated for different applications. Unlike chemical and biological methods, which have been extensively studied and applied, physical methods have received comparatively less attention in the literature. Our review uniquely fills this gap by providing an in-depth analysis of the mechanisms underlying each method, as well as exploring their diverse potential applications across various microorganisms and intracellular compounds. This review also offers critical insights into how these methods work at a fundamental level and the advantages and limitations of each technique, paying special attention to the conditions under which they excel. By integrating the latest research and technological advancements, we provide a comprehensive understanding on how these methods can be implemented in small and industrial scales. Ultimately, this review underscores the transformative potential of physical cell description techniques, positioning them as critical tools for advancing intracellular compound extraction in a sustainable, scalable, and cost-effective manner.

2. Physical Methods for Intracellular Compound Extraction

2.1. High-Pressure Homogenization

High-pressure homogenization (HPH) is a widely used method to disrupt cells for recovering intracellular materials [16]. A schematic diagram of high-pressure homogenizer is presented in Figure 1. In this process, a cell suspension is forced through a narrow valve under a high pressure, typically ranging from 10 to 300 MPa. When the cell suspension exits the valve, it experiences an intense pressure drop. As the velocity of the cell suspension changes, shear force is generated, breaking cell walls. High turbulence is generated by the high-speed flow through the narrow valve. Cavitation releases energy, creating mechanical damage to cell walls [21,22,23,24]. The combination of high turbulence, shear force, and cavitation breaks cells and releases intracellular contents. The cell disruption using high-pressure homogenization allows for the extraction and isolation of valuable substances from intracellular space. This method is efficient, scalable, and can be precisely controlled to maximize yield and preserve the functionality of the extracted compounds.

Many studies have reported using HPH to break microalgae cells for extracting various intracellular compounds (Table 1). For instance, high-pressure homogenization was used to break the cellular and chloroplast membranes of the microalgae Porphyridium cruentum to release B-Phycoerythrin, an intracellular pigment, and proteins [25]. The study explored various pressures and the number of homogenizations passes. A critical pressure point was identified at 100 MPa. Below this threshold, the chloroplasts membrane was only slightly damaged, resulting in a low B-Phycoerythrin and protein release. In contrast, pressures above 100 MPa effectively broke the cells, releasing significantly more B-Phycoerythrin. This is because pigments like B-Phycoerythrin are confined to chloroplasts, while other water-soluble proteins are also found in the cytoplasm. Operating at lower pressures may cause plasma membrane penetration, releasing proteins from the cytoplasm but not as effectively breaking down the chloroplasts. In the study of Zhang et al., HPH was applied to the microalgae Parachlorella kessleri, which is known for its high content of proteins, carbohydrates, and lipids, to recover intracellular compounds [26]. The microalgal suspension was passed through a high-pressure homogenizer at various pressures of 40, 80, and 120 MPa, for breaking cell walls. The study showed that the amount of protein released significantly increased with higher pressures and more passes through the high-pressure homogenizer. At 40 MPa, the protein concentration released increased from 250 to 919 mg/L after 10 passes. At 80 MPa, it rose from 500 to 2352 mg/L, and at 120 MPa, from 1375 to 3656 mg/L. In another study, HPH was demonstrated effective cell disruption for cells with recalcitrant cell walls, like Nannochloropsis microalgae, facilitating the extraction of proteins, lipids, and sugars [27]. The study found that the extraction efficiency of these compounds using high-pressure homogenization was affected by the age of microalgae cells since the cell wall thickness increased as the cell age. For microalgae, the pressure required for HPH varies based on cell wall thickness and composition. The cell walls of red algae, such as Porphyridium cruentum, are made of cellulose, xylan, or mannan fibrils and extensive matrix polysaccharides, making them particularly tough and thicker. In contrast, green algae like Parachlorella kessleri have cell walls primarily composed of cellulose, along with other proteins. This complexity of red algae requires a higher pressure to effectively disrupt red algae cell walls compared to green algae [28].

Besides microalgae, HPH has also been employed to extract intracellular compounds from yeast cells. In a study performed by Liu et al., high-pressure homogenizer was applied for breaking Saccharomyces cerevisiae yeast cells to release protein [29]. The study found that HPH at 80 MPa caused significant cell wall disruption and effectively extracted 50 μg intracellular protein from 1 g of yeast. In another study, Shynkaryk et al. investigated the potential synergistic effects of pulsed electric fields and high-voltage electric discharge with HPH to break Saccharomyces cerevisiae yeast cell walls [30]. The study found that high voltage electric discharge before HPH was effective for cell disruption. The shock waves generated by high voltage electric discharge impacted the physical integrity of the yeast cell walls, resulting in more disruption of the cells. In addition to the laboratory scale, high-pressure homogenizer was also tested at a pilot scale for extracting poly-β-hydroxybutyrate from recombinant Escherichia coli (E.coli) cells [31]. By using 55 MPa, the processes efficiently extracted poly-β-hydroxybutyrate (PHB) from the cell. With 20 L processing amount, the recovery rate reached 75% without chemical additives, while the green chemical assist extraction using sodium hypochlorite achieved a recovery rate of 80% and a purity of 95%. While HPH is suitable for a variety of cell types, the pressure required depends on the cell characteristics. Microalgae, with their thicker cell walls, demand a higher pressure input compared to bacterial and yeast cells. Yeast cell walls are primarily composed of a complex network of polysaccharides, mainly β-glucans along with chitin. Bacterial cell walls, on the other hand, consist mainly of peptidoglycan, making them less resistant to mechanical force. Therefore, among these three cell types, microalgae require the highest pressure for effective disruption, followed by yeast, with bacteria requiring the least [32].

Among an array of physical methods, HPH offers several significant advantages in intracellular compound recovery. One of the primary advantages of HPH is its high efficiency and yield. The technique generates mechanical forces—such as shear, cavitation, and turbulence—that are effective in breaking the cell walls of microorganisms, including tough cell types like bacteria, yeast, and algae. This leads to high recovery yields of intracellular compounds such as proteins, lipids, and enzymes, making HPH invaluable in industries like biotechnology, pharmaceuticals, and biofuels, where maximizing product recovery is crucial to ensuring cost-effective production. Another key benefit of HPH is its scalability. The technique can process large volumes of microbial cultures in a continuous operation, making it particularly well-suited for industrial-scale applications [33]. This ability to maintain efficiency even when scaled up is one of the reasons why HPH is favored in large-volume industries like food and beverage industry. For example, most milk is currently treated with HPH to enhance its microbial and physiochemical shelf life [34]. More importantly, HPH is highly versatile and able to break down diverse types of microbial cells, from bacteria and yeast to more resilient microalgae, through adjusting its pressure and number of passes. This versatility allows engineers to tune the process conditions (e.g., pressure, passes) to optimize extraction for different microorganisms.

Despite its clear advantages, high-pressure homogenization has several limitations that need to be addressed to fully optimize its performance. One of the primary drawbacks is heat generation. The mechanical forces involved in HPH generate significant amounts of heat, especially when high pressure forces are applied to viscosity liquids. The generated heat can elevate substrate temperature and potentially degrade heat-sensitive intracellular compounds, such as enzymes and essential oils. Although integrating cooling systems with HPH can partially solve this limitation, it adds the process complexity and cost to the process. Another limitation is the high energy consumption. Although HPH is relatively energy efficient at scale compared to other methods, operating HPH can be energy intensive, particularly at high pressure and with the multi-passes required for breaking thick cell walls like those of gram-positive bacteria and microalgae. One way to alleviate this challenge is to add an pretreatment, such as high-voltage electric discharge, to partially break cells before HPH, allowing HPH operate at low-pressure conditions [30]. Additionally, the shear forces generated during the process can fragment intracellular compounds or denature proteins [35].

Given its advantages and limitations, HPH is particularly well-suited for a number of practical applications in various industries. One of its key applications is the recovery of therapeutic proteins and enzymes from microbial cells like E. coli and yeast [36,37,38]. The ability of HPH to maintain the bioactivity of sensitive molecules while efficiently extracting them from cells makes it indispensable in the production of biologics, such as recombinant proteins and vaccines. In the biofuel industry, HPH is widely proposed to break the cell walls of microalgae to release lipid, which can be used to produce biodiesels [39,40]. Furthermore, HPH also plays in important role in the functional food industry, where it is used to extract bioactive compounds like proteins, enzymes, and vitamins from cultivated microbial cells.

Table 1. Summary of studies on high-pressure homogenization techniques extracting intracellular compound.

Microorganism	Target Compounds	Pressure	Recovery	Key Finding	Source
Porphyridium cruentum	B-Phycoerythrin and protein	270 MPa	Near 100%	100 MPa is a critical pressure point. Below the 100 MPa threshold, the chloroplast membrane was slightly damaged. Above 100 MPa, the cells were effectively broken, releasing significantly more B-Phycoerythrin.	[25]
Parachlorella kessleri	Proteins, carbohydrates	120 MPa/10 passes	3656 mg/L proteins	The concentration of released compounds significantly increases with higher pressures and more passes through the high-pressure homogenizer.	[41]
Nannochloropsis	proteins, lipids, and carbohydrates	125 MPa/6 passes	50.4 mg/g	The extraction efficiency of these compounds using high-pressure homogenization was affected by the age of microalgae.	[27]
Saccharomyces cerevisiae	Protein	80 MPa	50 μg protein/1 g of yeast	High-pressure homogenization resulted in almost complete damage of yeast cell walls.	[29]
Saccharomyces cerevisiae	bio-products	30–200 MPa	Near 100%	High-voltage electric discharge proved to have synergistic enhancement with high-pressure homogenizer.	[30]
Escherichia coli	poly-β-hydroxybutyrate	55 MPa	75%	The recovery rate reached 75% without chemical additives.	[31]

Table 1. Summary of studies on high-pressure homogenization techniques extracting intracellular compound.

Microorganism	Target Compounds	Pressure	Recovery	Key Finding	Source
Porphyridium cruentum	B-Phycoerythrin and protein	270 MPa	Near 100%	100 MPa is a critical pressure point. Below the 100 MPa threshold, the chloroplast membrane was slightly damaged. Above 100 MPa, the cells were effectively broken, releasing significantly more B-Phycoerythrin.	[25]
Parachlorella kessleri	Proteins, carbohydrates	120 MPa/10 passes	3656 mg/L proteins	The concentration of released compounds significantly increases with higher pressures and more passes through the high-pressure homogenizer.	[41]
Nannochloropsis	proteins, lipids, and carbohydrates	125 MPa/6 passes	50.4 mg/g	The extraction efficiency of these compounds using high-pressure homogenization was affected by the age of microalgae.	[27]
Saccharomyces cerevisiae	Protein	80 MPa	50 μg protein/1 g of yeast	High-pressure homogenization resulted in almost complete damage of yeast cell walls.	[29]
Saccharomyces cerevisiae	bio-products	30–200 MPa	Near 100%	High-voltage electric discharge proved to have synergistic enhancement with high-pressure homogenizer.	[30]
Escherichia coli	poly-β-hydroxybutyrate	55 MPa	75%	The recovery rate reached 75% without chemical additives.	[31]

2.2. Ultrasonication

Ultrasonication is emerging technique for extracting intracellular materials using ultrasound waves at frequencies between 20 kHz and 10 MHz. The ultrasound waves, generating through converting electrical energy into physical vibrations, transmit into a fluid and create pressure waves [42]. As these waves travel through a cell suspension, they generate compressible microbubbles that respond to the pressure waves. This phenomenon, known as cavitation, involves the microbubbles undergoing cycles of expansion and contraction [43,44]. Cavitation can occur in two forms: (1) stable cavitation at lower ultrasonic intensities, where bubbles oscillate without collapsing, and (2) inertial cavitation at higher intensities, where bubbles implode, causing significant biophysical effects (Figure 2). During the low-acoustic pressure cycles, stable cavitation is formed by microbubbles within the liquid. As the acoustic pressure increases, the cavitation bubbles collapse violently, generating intense physical, thermal, and chemical effects. These effects collectively contribute to cell disruption, membrane permeabilization, and the release of intracellular contents [45,46]. The severity of these effects depends on factors such as ultrasound intensity, frequency, and exposure time.

Ultrasonication has been used to disrupt diverse types of cells (Table 2). The study done by Liu et al. demonstrated that ultrasonication is effective to extract intracellular protein from yeast [14]. They found that increased acoustic power and duty cycles enhanced cell disruption and protein release. The study also compared horn-type sonication to bath-type sonication for yeast cell disruption. A horn-type sonicator consists of a probe that directly contacts the sample and transmits ultrasonic energy into the medium, while a bath-type sonicator generates ultrasonic waves within a tank filled with liquid (usually water). The sample is placed in a container, which is submerged in the bath. The ultrasonic waves are transmitted through the liquid, creating cavitation in the sample container indirectly. They revealed that horn-typed sonication is more effective to disrupt yeast cells. In another study, Wu et al. investigated the mechanisms of ultrasonic disruption in yeast cells (Saccharomyces cerevisiae), analyzing how different processing parameters impact the release of cell wall polysaccharides and intracellular proteins [47]. Their study revealed that at low acoustic intensities (10 W/cm²), cell wall disruption occurs more rapidly, resulting in a quicker release of cell wall polysaccharides compared to intracellular proteins. However, at higher intensities (24 and 39 W/cm²), this pattern reverses, with proteins being released more rapidly than polysaccharides. Moreover, an increased processing temperature, more cell wall polysaccharides but few intracellular proteins were released. The findings suggested that ultrasonic cell disruption initiates with the breakdown of the cell wall, followed by the cell membrane.

Ultrasonication has also been applied to disrupt microalgae cells. A study investigated how ultrasonic intensity, duration, temperature, culture time, and ethanol concentration affected the extraction of polysaccharides from Chlorella pyrenoidosa [48]. The researchers optimized the extraction process using an orthogonal design and found that the highest polysaccharide yield, 44.8 g/kg, was achieved with an ultrasound intensity of 400 W ultrasound for 800 s, followed by incubation at 100 °C for 4 h in 80% ethanol. The study also presented a straightforward method for isolating polysaccharides from the crude extracts using column chromatography purification. Aouira et al. investigated the extraction of phycobiliproteins from microalgae Spirulina platensis using ultrasound technologies [49]. The study compared the yield and purity of phycobiliproteins extracted by ultrasound to those obtained using the pulsed electric field method. The pulsed electric field method, conducted at 38 kV/cm for 0.03 s, resulted in the highest yields of phycocyanin (0.084 g/L), allophycocyanin (0.065 g/L), and phycoerythrin (0.024 g/L). In contrast, ultrasound technology achieved similar yields by treating the samples at 35 kHz, 750 W for 90 min. Despite producing comparable yields, ultrasound demanded significantly longer treatment and higher energy input, making it a less efficient and less preferable method for extracting phycobiliproteins from Spirulina platensis compared to the pulsed electric field method.

In addition to extracting polysaccharides and proteins, ultrasonication can also be used to extract intracellular lipid. Natarajan et al. applied continuous ultrasonication to disrupt two marine microalgae species for lipid extraction [50]. The authors found that fatty acids release patterns differed between different microalgae species. Lipids in microalgae with rigid cell walls were easily released to the liquid after cell disruption, whereas lipids in microalgae having flexible cell membranes tend to be retained on membranes after disruption. In general, cell disruption and lipid release efficiencies correlate with ultrasound energy consumption. In the Ronald Halim et al. study, the researchers demonstrated that the ultrasonication effectively extracted all available intracellular lipids [51]. Their result also indicated that higher initial cell concentration promoted faster spread of the shock wave generated by the microbubble implosion but reduced the amount of energy received by each cell. When the initial cell concentration was low, the effect of increasing the transmission rate was dominant. However, beyond a certain threshold, the effect of the energy reduction outweighs the effect of the increase in the transmission rate. Lipids recovered from disrupted microalgae cells with 30 min of ultrasonication were found to have similar triglyceride profiles, but the higher degree of unsaturation (61.6 wt% of triglyceride had more than eight double bonds) compared to the lipids in intact microalgal cells (24.4 wt% of triglyceride had more than eight double bonds). A possible explanation for the changes in triglyceride composition could be the liberation of neutral lipids located inside cells or associated with membranes.

Ultrasonication offers distinct advantages for extracting various intracellular compounds, particularly in small- to medium-scale applications where control and precision are essential. One of the main strengths of ultrasonication is its tunability (e.g., adjusting frequency, intensity, duration, and configurations) to achieve selective and controlled cell disruption through the phenomenon of acoustic activation [47]. This high-level of tunability is not seen in other physical methods, such as HPH and mechanical bead milling. Moreover, the localized shear forces generated during cavitation can effectively break down microbial cell walls while preserving the integrity of sensitive intracellular compounds like proteins, enzymes, and nucleic acids. This makes ultrasonication especially applicable in the high-value products, such as pharmaceuticals and nutraceuticals, where the preservation of bioactivity is critical. In addition, although ultrasound generates heat during cavitation, the heat generation is localized and relatively mild compared with HPH and mechanical bead milling, which generates significant heat due to mechanical force. This makes the ultrasound method more suitable for heat-sensitive and high-value compounds.

Scalability is perhaps the biggest challenge of the ultrasound method in academia and industry. While the ultrasound method excels at small- and medium-scale processing, it becomes less efficient in large-scale processing [52]. As the volume of liquid medium increases, the energy of the sound waves attenuates, leading to incomplete or uneven cell disruption, especially for those cells far away from the ultrasound generator. Another known limitation of ultrasonication is that it is less effective to process high-viscosity liquids. When the viscosity of the medium increases, the ability of sound waves to propagate uniformly is hindered. This reduced wave propagation dampens the formation of cavitation bubbles, which are essential for cell disruption. Consequently, cavitation occurs less uniformly, resulting in lower mechanical forces and less efficient cell breakage [53]. In addition, it was reported that the ultrasound-induced cavitation can generate free radicals in the liquid medium [54,55], which may cause undesirable oxidative reaction between the free radicals and intracellular compounds, thus degrading the product quality.

Overall, ultrasound is particularly suitable for small- to medium-scale applications in the industries like functional foods and pharmaceuticals, where controlled, gentle cell disruption is necessary to keep the integrity and bioactivity of the targeted compounds. Moreover, users need to be particularly careful about using the ultrasound method to process high-viscosity liquid medium. If needed, mechanical stirring and dilution can be combined with ultrasonication to alleviate the viscosity effect.

Table 2. Summary of studies on ultrasonication techniques extracting intracellular compound.

Microorganism	Target Compounds	Key Finding	Source
Saccharomyces cerevisiae	Protein	The intracellular protein release followed first-order kinetics. The horn-type sonication was proved more effective than bath-type sonication for yeast cell disruption.	[14]
Saccharomyces cerevisiae	Polysaccharides, proteins	At low acoustic intensities, the release of cell wall polysaccharides was quicker. At higher intensities proteins, were released more rapidly than polysaccharides.	[47]
Chlorella pyrenoidosa	Polysaccharides	The highest polysaccharide yield, 44.8 g/kg, was achieved with an ultrasound intensity of 400 W ultrasound for 800 s, followed by incubation at 100 °C for 4 h in 80% ethanol.	[48]
Spirulina platensis	Phycobiliproteins	Ultrasound required significantly longer treatment times and higher energy input, making it less efficient and less preferable for extracting phycobiliproteins compared to the pulsed electric field method.	[49]
Marine microalga	Fatty acids	The fatty acid release pattern varied among different microalgae species.	[50]
Microalga	Lipids	Higher initial cell concentration promoted faster spread of the shock wave generated by the microbubble implosion but reduced the amount of energy received by each cell.	[51]

Table 2. Summary of studies on ultrasonication techniques extracting intracellular compound.

Microorganism	Target Compounds	Key Finding	Source
Saccharomyces cerevisiae	Protein	The intracellular protein release followed first-order kinetics. The horn-type sonication was proved more effective than bath-type sonication for yeast cell disruption.	[14]
Saccharomyces cerevisiae	Polysaccharides, proteins	At low acoustic intensities, the release of cell wall polysaccharides was quicker. At higher intensities proteins, were released more rapidly than polysaccharides.	[47]
Chlorella pyrenoidosa	Polysaccharides	The highest polysaccharide yield, 44.8 g/kg, was achieved with an ultrasound intensity of 400 W ultrasound for 800 s, followed by incubation at 100 °C for 4 h in 80% ethanol.	[48]
Spirulina platensis	Phycobiliproteins	Ultrasound required significantly longer treatment times and higher energy input, making it less efficient and less preferable for extracting phycobiliproteins compared to the pulsed electric field method.	[49]
Marine microalga	Fatty acids	The fatty acid release pattern varied among different microalgae species.	[50]
Microalga	Lipids	Higher initial cell concentration promoted faster spread of the shock wave generated by the microbubble implosion but reduced the amount of energy received by each cell.	[51]

2.3. Mechanical Milling

Mechanical milling (also referred to as ‘beating’ in some sources) is a simple and effective method for breaking cell walls to release intracellular compounds. This technique applies mechanical forces through the use of small, hard beads (usually made of steel, ceramic) to break down cell structures [56]. Two typical pieces of milling equipment used to disrupt cells are bead mills and ball mills. In both pieces of equipment, cells are disrupted by grinding them between beads or balls within a closed container. The beads or balls agitate and collide with the cells, causing physical shear forces that break apart the cell walls and release their contents. The radial acceleration of beads or balls results in different velocity generating high shear forces (Figure 3) [57]. In bead mills, kinetic energy is transferred to the grinding media by stirring shafts in vertical or horizontal configurations. Depending on the intensity of the stress applied, different particle size reduction mechanisms can occur. At low stress, surface abrasion leads to the removal of fine particles from the parent particles, resulting in most of the particles being close in size to the parent particles. At relatively intense stress, particle cleavage occurs, producing fragments slightly smaller than the parent particles. At rapidly intense stress, fracture leads to the production of small particles with a wide particle size distribution [58]. Ball milling, on the other hand, operates within a rotating container where the balls generate forces including impact, abrasion, and corrosion forces. Impact results from repeated high-energy impacts causing surface cracking. Corrosion wear involves galvanic interactions between the cell and the balls or between different abraded and unabraded points on the balls’ surface [59]. When cells are crushing and grinding in the milling, the ball size distribution, and rotation speed both determine disintegration ability [60,61]. Between the two, bead mills are generally the preferred choice because of their greater efficiency in breaking down microbial cells due to their smaller bead size generating higher shear force. Overall, the milling method offers several advantages, including continuous operation, relatively low capital costs, and suitability for applications ranging from the laboratory scale to the industrial scale [60,62].

Milling technology has been used to disrupt bacterial cell walls. Tamer et al. applied a continuous flow high-speed bead mill to recover intracellular poly(b-hydroxybutyric acid) (PHB) from Alcaligenes latus cells [63]. Without any chemical involved, an 85% loaded bead achieved over a 50% improvement compared to an 80% loaded bead, which had a disruption rate constant of 0.44. The results also indicated that PHB purity increased with the number of passes. However, extreme physical disruption could result in a micronization phenomenon occurring to PHB, resulting in loss of PHB. Although bead milling alone does not achieve higher extraction efficiency than solvent extraction, it offers better energy efficiency since highly concentrated slurries can be processed in bead mills. Another study done by the same group studied how different bead milling parameters affect the protein extraction from Alcaligenes latus cells [18]. Their study showed that the mean diameter of the beads (512 to 945 µm) did not affect the disruption rate of Alcaligenes latus cells, since the bacterial cell size was too small (0.5–1.0 µm in diameter). With a mean diameter of 512-µm beads, complete disruption was achieved by the eighth pass at an 85% bead loading, whereas a reduced loading of 75% did not release all the cellular protein even after 16 passes. The biomass concentration was not a dependent factor for bead mill disruption effectiveness. Another study investigated the extraction of intracellular enzymes from Arthrobacter sp. DSM 3747 by examining the effects of various operational parameters of the stirred ball mill on disintegration results [60]. They found that, under the same operating conditions, achieving a disintegration rate of 50% with 1.28 mm beads requires 20 times higher energy compared to smaller beads of 0.2 mm. The optimized operational parameters combination was small grinding beads (0.3 mm) with a moderate-to-high cell concentration in the suspension (about 50%) and operating at low-to-moderate agitator speeds (6 m/s circumferential speed of agitator disks).

In addition, bead milling was applied to algae for cell disruption. A study investigated the optimization of bead milling for microalgae cell disruption, focusing on how different bead sizes affect the process efficiency, energy consumption, and the release of proteins and carbohydrates [64]. The result indicated that smaller bead size, which induces more shearing forces, improved extraction yield with a lower energy consumption. Specifically, the bead size range of 0.3–0.4 mm was determined as optimal bead size for balancing cell disruption efficiency and energy consumption for microalgae. In another study, researchers employed bead milling to disrupt cells for isolating proteins from the green microalga Tetraselmis sp. [65]. The microalgae were milled by bead milling to release proteins, followed by centrifugation and ion exchange chromatography to purify the proteins. The resulting algae-soluble isolate comprised 64% protein and had a clear appearance with 100% solubility at pH above 5.5, making it a potential food ingredient. In a study, Loureiro et al. studied five cell-disruption methods including freeze–thaw cycles, bead milling, high-speed homogenization, microwave, and sonication to disrupt microalgae (Coelastrella sp.) for releasing intracellular biochemical compounds [66]. They found that sonication showed the highest efficiency in the release of proteins, carbohydrates, and lipids, followed by bead milling. However, considering the balance of the extraction efficiency and energy consumption, bead milling can be considered the most efficient method. Another study examined the effects of nitrogen depletion on bead-milling performance for releasing cellular components from Neochloris oleoabundans [67]. Under the same bead-milling operation condition, the study revealed that nitrogen-depleted cells had significantly higher release rates of biomass and biochemical components, with a release ratio of 0.57, compared to 0.38 for nitrogen-replete cells. Nitrogen-depleted conditions enhanced the release intracellular compounds, achieving up to 68% carbohydrate, 59% protein, and 56% lipids (w/w) release. Additionally, the energy consumption per kilogram of released product was lower for nitrogen-depleted cells, indicating more efficient cell disruption.

Several studies examined the disruption of yeast cells using bead milling to release glucose 6-phosphate dehydrogenase. One study used a vertical bead mill to extract glucose 6-phosphate dehydrogenase [68]. Their results identified 2300 rpm for 6 min as optimal conditions for yeast disruption, achieving higher proportion of glucose 6-phosphate dehydrogenase release in total proteins without heat denaturation. Another study used a horizontal bead mill in continuous recycle mode to extract glucose 6-phosphate dehydrogenase [69]. The result indicated that optimal total protein release (37.3 mg/mL) was achieved with a 24 h/L feeding rate and 50% (w/v) initial biomass concentration. However, a higher biomass concentration loading generated heat, which decreased glucose 6-phosphate dehydrogenase activity. To balance optimal yield, minimal glucose 6-phosphate dehydrogenase degradation, and manageable viscosity during afterward centrifugation, the study concluded that a 20% (w/v) biomass load was optimal. This reduced heat generation and maintained enzyme bioactivity while facilitating easier separation after milling.

Milling has been demonstrated to be useful in extracting proteins and lipids from various cell types (Table 3). It has particularly advantageous for tough-walled cells, such as microalgae, due to the high mechanical forces generated by the grinding media, which can effectively disrupt rigid cell walls with relatively low energy consumption [70]. Additionally, milling systems are scalable from laboratory to industrial scale, and bead milling can be adapted for continuous processing of viscous and highly concentrated cell suspensions, making it beneficial for industrial applications [71]. In addition, mechanical milling provides consistent and uniform cell disruption, which is critical for maximizing the recovery of intracellular compounds.

However, mechanical milling methods come with certain limitations. One primary concern is the significant heat generation due to the friction between beads/balls during the milling process [72,73]. The generated heat is difficult to remove from the container and it could damage heat-sensitive intracellular compounds. This requires careful management of heat removal by additional cooling system. Moreover, compared to other physical methods like ultrasonication or high-pressure homogenization, mechanical milling provides less precise control over the disruption process, potentially leading to the co-extraction of unwanted cell debris, complicating downstream purification processes. Additionally, there is a risk of contamination from the grinding media, as the beads and balls can wear down over time due to the mechanical forces of impact, abrasion, and attrition, releasing particles into the samples. For applications requiring high purity, such as pharmaceuticals or fine chemicals, this contamination can be problematic and may require additional filtration or purification steps [59].

Overall, due to its versability, mechanical milling is widely used in industrial-scale bioprocessing, particularly for breaking tough cell types like yeast, microalgae, and fungi. Its scalability and cost-effectiveness make it suitable for large-scale operations where high throughput and uniform cell disruption are critical. However, due to its high heat generation, it is less ideal for recovering thermolabile compounds, where more precise methods like ultrasonication may be preferred.

Table 3. Summary of studies on milling techniques extracting intracellular compound.

Microorganism	Target Compounds	Recovery	Parameters	Key Finding	Source
Alcaligenes latus	poly(b-hydroxybutyric acid)	44% disruption rate,1.2 kg PHB/kg pellet	512 µm mean bead diameter, 80% bead loading	The extreme mechanical disruption could result in micronization phenomenon to poly(b-hydroxybutyric acid).	[63]
Alcaligenes latus	Proteins	Near 100%	512 to 945 µm bead diameter, 85% bead loading	The mean diameter of the beads did not affect the disruption rate of Alcaligenes latus cells. Biomass concentration did not influence the effectiveness of bead mill disruption.	[18]
Arthrobacter sp.	Proteins	50% disintegration rate	300 µm bead diameter, 80% dead loading	Cell disruption correlates with energy input.	[60]
microalgae	Proteins and carbohydrates	30–50% proteins, 10–30% carbohydrates	300–400 µm bead diameter, 65% bead loading	The bead size range of 0.3–0.4 mm was determined as optimal balancing cell disruption efficiency and low energy consumption.	[64]
Tetraselmis sp.	Proteins	64% proteins	400–600 µm bead diameter, 65% bead loading	Bead milling followed by centrifugation and ion exchange chromatography resulted algae-soluble isolate comprised 64% protein and 100% solubility at pH levels above 5.5.	[65]
Coelastrella sp.	biochemical compounds	94% disruption efficiency	500 µm bead diameter, 32% bead loading	Considering the balance of the extraction efficiency and energy consumption, bead milling can be considered the most efficient method.	[66]
Neochloris oleoabundans	Carbohydrate, protein, and lipids	68% carbohydrate, 59% protein, and 56% lipids	400–600 µm bead diameter	Nitrogen depletion can enhance the cost-effectiveness of microalgal biomass processing by bead milling.	[67]
Saccharomyces cerevisiae	glucose 6-phosphate dehydrogenase	15 mg/mL protein	500 µm bead diameter	The vertical bead mill at 2300 rpm for 6 min was optimal conditions for yeast disruption and glucose 6-phosphate dehydrogenase release without heat denaturation.	[68]
Saccharomyces cerevisiae	glucose 6-phosphate dehydrogenase	37.3 mg/mL protein	300 µm bead diameter	In a horizontal bead mill operating in continuous recycle mode, the optimal total protein release was achieved with a 24 h/L feeding rate and a 50% (w/v) initial biomass concentration.	[69]

Table 3. Summary of studies on milling techniques extracting intracellular compound.

Microorganism	Target Compounds	Recovery	Parameters	Key Finding	Source
Alcaligenes latus	poly(b-hydroxybutyric acid)	44% disruption rate,1.2 kg PHB/kg pellet	512 µm mean bead diameter, 80% bead loading	The extreme mechanical disruption could result in micronization phenomenon to poly(b-hydroxybutyric acid).	[63]
Alcaligenes latus	Proteins	Near 100%	512 to 945 µm bead diameter, 85% bead loading	The mean diameter of the beads did not affect the disruption rate of Alcaligenes latus cells. Biomass concentration did not influence the effectiveness of bead mill disruption.	[18]
Arthrobacter sp.	Proteins	50% disintegration rate	300 µm bead diameter, 80% dead loading	Cell disruption correlates with energy input.	[60]
microalgae	Proteins and carbohydrates	30–50% proteins, 10–30% carbohydrates	300–400 µm bead diameter, 65% bead loading	The bead size range of 0.3–0.4 mm was determined as optimal balancing cell disruption efficiency and low energy consumption.	[64]
Tetraselmis sp.	Proteins	64% proteins	400–600 µm bead diameter, 65% bead loading	Bead milling followed by centrifugation and ion exchange chromatography resulted algae-soluble isolate comprised 64% protein and 100% solubility at pH levels above 5.5.	[65]
Coelastrella sp.	biochemical compounds	94% disruption efficiency	500 µm bead diameter, 32% bead loading	Considering the balance of the extraction efficiency and energy consumption, bead milling can be considered the most efficient method.	[66]
Neochloris oleoabundans	Carbohydrate, protein, and lipids	68% carbohydrate, 59% protein, and 56% lipids	400–600 µm bead diameter	Nitrogen depletion can enhance the cost-effectiveness of microalgal biomass processing by bead milling.	[67]
Saccharomyces cerevisiae	glucose 6-phosphate dehydrogenase	15 mg/mL protein	500 µm bead diameter	The vertical bead mill at 2300 rpm for 6 min was optimal conditions for yeast disruption and glucose 6-phosphate dehydrogenase release without heat denaturation.	[68]
Saccharomyces cerevisiae	glucose 6-phosphate dehydrogenase	37.3 mg/mL protein	300 µm bead diameter	In a horizontal bead mill operating in continuous recycle mode, the optimal total protein release was achieved with a 24 h/L feeding rate and a 50% (w/v) initial biomass concentration.	[69]

2.4. Pulsed Electric Field

Pulsed electric field is an emerging and non-thermal method used for intracellular compound extraction, primarily in the biotechnology, food, and pharmaceutical industries. It disrupts cell walls or membranes by applying short and high-voltage electrical pulses (10–80 kVcm⁻¹), creating temporary or irreversible pores or gaps in the cell membrane, allowing molecules, ions, or other substances to pass through that would normally be restricted [74]. The electrical pulses alter the transmembrane potential, causing the formation of nanopores in the cell membrane’s phospholipid layer. These nanopores arise from the reorganization and destabilization of lipid molecules induced by the electric field. This process, known as electroporation or electropermeabilization, is influenced by several key electrical parameters, including the field strength, pulse shape and duration, number of pulses, and specific energy used in treatment [75]. As shown in Figure 4, this electroporation can be reversible or irreversible based on the strength of the applied electrical field. When the applied external electrical field (E_e) is higher than the critical electrical field strength of the cell membrane (E_c), membrane permeabilization takes place but the cell membrane can recover after the exposure is performed. When the E_e further increases, the E_e is much higher than E_c to the certain point that the permeabilization is not able to be recovered. The release of intracellular substance resulting from pulsed electric field is a relatively gentle cell-disintegration process, typically performed at ambient temperatures and without introducing additional impurities [76]. The mild treatment avoids heat degradation of the targeted intracellular materials while releasing them. Additionally, the disruption process is rapid, occurring within a few seconds.

The pulsed electric field made the cell membrane permeable, as demonstrated by the percentage propidium iodine fluorescent dye up taken by cells, which can only pass through the non-intact membrane. Luengo et al. examined the impact of pulsed electric field treatments on the permeabilization of Chlorella vulgaris and the extraction of pigments [77]. The study found that after 150 µs of pulsed electric field treatment, it caused irreversible electroporation resulting in 100% cell death of Chlorella vulgaris. The application of 20 kVcm⁻¹ for 75 µs increased pigment extraction yields by 1.2 to 2.1 times after 1 h incubation compared to immediate extraction, which were 1100 µg carotenoids, 2500 µg chlorophylle a, and 1100 µg chlorophylle b per g of culture. In another study, Lam et al. investigated the release of protein from microalgae using two sets of pulsed electric field: (i) batch pulsed electric field with electric field strength between 7.5 and 30 kV cm⁻¹, and (ii) continuous flow pulsed electric field treatment with electric field strength of 20 kV cm⁻¹ with 13 mL min⁻¹ flow rate [19]. They found that with increased pulsed electric field energy, the relative ion release increased, indicating that the pulsed electric field made the microalgae membrane more permeable. However, a low protein yield was obtained as intracellular proteins remained entrapped. One study examined how the pulsed electric field treatment energy and electric filed strength and biomass concentration affected cell disruption using the microalgae Auxenochlorella protothecoides [76]. It was observed that the disruption efficiency increased as specific treatment energy increased, while the field strength had a minimal impact. For pigments and proteins extraction from Parachlorella kessleri microalgae, high-voltage electrical discharges were found effective for extracting ionic components and carbohydrates but less so for proteins and pigments, achieving a maximum protein extraction of only 750 mg/L, which was about 15% of the total protein content [26].

Coustets et al. developed a pulsed electric field-based process to extract cytoplasmic proteins from three microalgae species: Nannochloropsis salina, Chlorella vulgaris, and Haematococcus pluvialis [78]. The study examined various electric field strengths, pulse durations, and incubation conditions, finding that optimal extraction conditions were species-dependent. Nannochloropsis salina required a stronger electric field (6 kV cm⁻¹) for effective release of 25 µg protein/mL culture. For Chlorella vulgaris and Haematococcus pluvialis, milder conditions (4.5 kV cm⁻¹) sufficed to release 30 µg protein/mL culture due to their larger cell sizes. Nannochloropsis salina, with higher field strength needed, had extraction improved significantly after a second cycle of 15 bipolar pulses, while Chlorella vulgaris did not benefit from an additional cycle. The study also demonstrated a linear relationship between cell concentration and extracted protein concentration, indicating that higher cell densities directly increased the amount of protein extracted. Postma et al. observed a synergistic effect between pulsed electric field and temperature treatments to disrupt microalgae Chlorella vulgaris [79]. They demonstrated that applying pulsed electric field at temperatures between 25 °C and 65 °C increased cell permeability and selectively enhanced the release of carbohydrates, achieving up to 39% carbohydrate extraction while retaining over 95% of the proteins within the cells. This synergistic effect was most pronounced at 55 °C, particularly for carbohydrate release. The combined pulsed electric field and temperature treatment effectively released small, water-soluble molecules but was less efficient for protein extraction compared to bead milling. Luengo et al. compared the effects of millisecond- and microsecond-pulsed electric field treatments on the permeabilization and extraction of pigments from microalgae Chlorella vulgaris [80]. The study found that permanently electroporation occurred at 4 kV cm⁻¹ with millisecond pulses, while microsecond pulses required 10 kV cm⁻¹. Electroporation effectiveness was linked to pulse duration and frequency: millisecond pulses needed 20 pulses at 5 kV cm⁻¹, whereas microsecond pulses required shorter treatment times but higher electric field strengths (≥15 kV cm⁻¹). To achieve a similar degree of permanent electroporation, a reduction in pulse duration from milliseconds to microseconds required a three-fold increase in electric field strength. For effective pigment extraction, microsecond pulses proved more energy-efficient, consuming 30 kJ/L compared to 150 kJ/L for millisecond pulses. Additionally, microsecond pulses (15 kV cm⁻¹, 25 pulses) yielded higher pigment extraction, with concentrations of carotenoids and chlorophylls a and b at 1.09, 3.95, and 2.17 mg/L of culture, respectively. In contrast, millisecond pulses (5 kV cm⁻¹, 20 pulses) resulted in yields of 1.06, 2.90, and 1.69 mg/L of culture for the same pigments. These findings indicate that microsecond pulses offer a more energy-efficient and effective method for pigment extraction from microalgae.

Studies were also conducted to apply pulsed electric field for extracting protein from Saccharomyces cerevisiae, which is rich in intracellular proteins and other bioactive compounds. Ganeva et al. assessed the effect of medium pH on the protein release from yeast by pulsed electric field treatment [81]. The result indicated that the more alkalic condition increased the intracellular protein release. Cell concentration also influenced protein release efficiency—a higher cell concentration reduces optimal field strength. About 90% of the total soluble protein release occurred only after dilution and incubation of the permeabilized cells in buffer with pH 8–9. Fincan et al. explored the efficacy of pulsed electric field treatment in enhancing the extraction of red pigment from red beetroot [82]. By applying 270 rectangular pulses at 1 kV cm⁻¹, the research achieved approximately 90% total red coloring and ionic content release after 1 h aqueous extraction. It offers a low-energy alternative for the extraction of valuable compounds from plant tissues. The pulsed electric field treatment significantly led to a higher release of pigment and ionic species into the solution, compared to the traditional freezing and thawing method.

One of the major advantages of pulsed electric field is its gentle, non-invasive nature. Unlike mechanical milling or HPH, pulsed electric field does not generate a large amount of heat due to friction, making it ideal for extracting heat-sensitive compounds, such as pigments enzymes, and functional proteins (Table 4). Another clear advantage of pulsed electric field is its selectivity. It selectively disrupts the cell member without causing extensive damage to other cellular components, thus enhancing the product purity and simplifying downstream purification by reducing the co-extraction of undesirable cell debris. In addition, with operation times ranging from microseconds to milliseconds, pulsed electric field is a rapid and efficient method. Additionally, the process can be precisely controlled.

Pulsed electric field has some challenges to overcome. First, unlike mechanical milling, which can be universally used with similar operations, the pulsed electric field technique depends heavily on the optimization of process parameters like electric field strength, pulse duration, and the conductivity of the medium for each type of microbial cells and liquid medium. Failing to optimize these parameters will likely cause incomplete extraction of intracellular compounds or excessive cell damage. Second, although this technology is highly scalable to the industrial level, it requires specialized equipment, which usually leads to high capital investment to purchase and install the equipment and auxiliary facility to initiate the process. Additionally, the viscosity of the suspension can impact the uniformity of the electric field. In high-density broths or non-conductive media, cells may not experience uniform field exposure, leading to incomplete disruption [74].

As an emerging technology, the pulsed electric field technology is still largely in the research and pilot-scale stage for many applications, although there are some industrial uses for the purpose of extending shelf-life of liquid foods (like fruit juices). The motioned challenges, including the need for process optimization, specialized equipment, and limitation with breaking down tough cell types like fungi, have slowed the wide industrial adoption of this technique for intracellular extraction. However, there is a tremendous interest in commercializing this technique for the recovering of high-value protein and enzymes from microbial cells in the pharmaceutical and biotechnology industry, due to its capability to preserve bioactivity and ensure purity of the target compounds.

Table 4. Summary of studies on pulsed electric field techniques extracting intracellular compound.

Microorganism	Target Compounds	Electrical Field Strength	Key Finding	Source
Chlorella vulgaris	Carotenoids and Chlorophylls a and b	20 kVcm−1	After 150 µs of pulsed electric field treatment, it caused irreversible electroporation resulting the Chlorella vulgaris cell 100% death.	[77]
Chlorella vulgaris and Neochloris oleoabundans	Protein	20–30 kVcm−1	Increased pulsed electric field energy enhanced membrane permeability in microalgae. However, protein yield remained low, as intracellular proteins were still trapped.	[19]
Auxenochlorella protothecoides	Intracellular valuables	23–43 kVcm−1	The efficiency of disruption increased as specific treatment energy increased, while the field strength had minimal impact.	[76]
Parachlorella kessleri	Protein and pigment	40 kVcm−1	High-voltage electrical discharge was found effective for extracting ionic components.	[26]
Nannochloropsis salina, Chlorella vulgaris, and Haematococcus pluvialis	Protein	4.5 and 6 kVcm−1	A linear relationship between cell concentration and extracted protein concentration was found. Optimal extraction conditions were species dependent.	[78]
Chlorella vulgaris	Protein and carbohydrates	17.1 kVcm−1	A synergistic effect between pulsed electric field and temperature treatments increased cell permeability and released small, water-soluble molecules.	[79]
Chlorella vulgaris	Pigments	5–15 kVcm−1	Microsecond pulses offer a more energy-efficient and effective method than millisecond pulses for pigment extraction from microalgae.	[80]
Saccharomyces cerevisiae	Protein	2.5–5.5 kVcm−1	More alkalic conditions increased the intracellular protein release. A higher cell concentration reduced optimal field strength.	[81]
Red beetroot	Red pigment	1 kVcm−1	Pulsed electric field treatment significantly led to a higher release of pigment and ionic species into solution, compared to traditional freezing and thawing method.	[82]

Table 4. Summary of studies on pulsed electric field techniques extracting intracellular compound.

Microorganism	Target Compounds	Electrical Field Strength	Key Finding	Source
Chlorella vulgaris	Carotenoids and Chlorophylls a and b	20 kVcm−1	After 150 µs of pulsed electric field treatment, it caused irreversible electroporation resulting the Chlorella vulgaris cell 100% death.	[77]
Chlorella vulgaris and Neochloris oleoabundans	Protein	20–30 kVcm−1	Increased pulsed electric field energy enhanced membrane permeability in microalgae. However, protein yield remained low, as intracellular proteins were still trapped.	[19]
Auxenochlorella protothecoides	Intracellular valuables	23–43 kVcm−1	The efficiency of disruption increased as specific treatment energy increased, while the field strength had minimal impact.	[76]
Parachlorella kessleri	Protein and pigment	40 kVcm−1	High-voltage electrical discharge was found effective for extracting ionic components.	[26]
Nannochloropsis salina, Chlorella vulgaris, and Haematococcus pluvialis	Protein	4.5 and 6 kVcm−1	A linear relationship between cell concentration and extracted protein concentration was found. Optimal extraction conditions were species dependent.	[78]
Chlorella vulgaris	Protein and carbohydrates	17.1 kVcm−1	A synergistic effect between pulsed electric field and temperature treatments increased cell permeability and released small, water-soluble molecules.	[79]
Chlorella vulgaris	Pigments	5–15 kVcm−1	Microsecond pulses offer a more energy-efficient and effective method than millisecond pulses for pigment extraction from microalgae.	[80]
Saccharomyces cerevisiae	Protein	2.5–5.5 kVcm−1	More alkalic conditions increased the intracellular protein release. A higher cell concentration reduced optimal field strength.	[81]
Red beetroot	Red pigment	1 kVcm−1	Pulsed electric field treatment significantly led to a higher release of pigment and ionic species into solution, compared to traditional freezing and thawing method.	[82]

Physical cell disruption methods include high-pressure homogenization, ultrasonication, milling, and pulsed electric fields. These methods can also be combined with chemical and biological approaches. Each technique has its own advantages and disadvantages, and their application depends on the target compounds and specific processing requirements (Figure 5).

3. Physical Assisted Methods

Combining physical techniques with chemical and biological methods enhances extraction efficiency by reducing energy consumption, improving selectivity, and minimizing damage to sensitive compounds. The use of enzymes or mild chemical solvents alongside physical techniques allows physical forces to more easily complete the disruption, thus reducing the energy required for physical techniques. Each method brings unique advantages, and when combined, they create a synergistic effect that improves overall extraction yields (Table 5).

3.1. Combination of Physical with Chemical Methods

The combination of physical and chemical methods to extract intracellular compounds offers a promising avenue for enhancing the efficiency and yield of biologically active substances from various biological matrices. By integrating physical methods like ultrasonication or homogenization with chemical treatments such as solvent extraction, this hybrid approach can effectively disrupt cell walls and membranes, facilitating deeper penetration of chemicals that solubilize target compounds.

Zhang et al. investigated the efficacy of combining different physical treatments—pulsed electric fields, high-voltage electrical discharge, and ultrasonication—with green solvent extraction to extract water-soluble molecules (carbohydrates and proteins) and water-insoluble molecules (chlorophyll a) from three different microalgal species [83]. The research revealed that high-voltage electrical discharge-assisted solvent extraction was most effective for extracting carbohydrates, while ultrasonication-assisted solvent extraction was more efficient for extracting proteins and chlorophyll a from microalgal species (Nannochloropsis sp., P. tricornutum, and P. kessleri). Combining ultrasound with solvent extraction has proven effective in enhancing the release of intracellular compounds. For instance, a study led by Monks et al. explored methods of cell disruption to optimize the extraction of carotenoids from fungi [84]. Their results demonstrated that ultrasound alone showed relatively low carotenoids recovered while combining ultrasound with sodium bicarbonate doubled the number of recovered carotenoids. Another study led by Zheng et al. examined the use of ultrasonic-assisted deep eutectic solvent extraction to extract phenolic compounds from foxtail millet bran [85]. They optimized a green extraction method to maximize phenolic content. The optimized conditions involved a deep eutectic solvent composed of betaine and glycerol in a 1:2 molar ratio and ultrasonic power at 247 W. Under the optimal condition, a total phenolic content of 7.80 mg ferulic acid equivalent per gram was achieved, yielding higher total phenolics, flavonoids, antioxidant activity, and acetylcholinesterase inhibitory activity compared to conventional solvent extraction methods. Scanning electron microscopy revealed significant microstructural disruption in the millet bran, with more pores and cracks, after ultrasonic-assisted deep eutectic solvent extraction. To evaluate the effects of ultrasound-assisted lipid extraction using solvents from wet microalgal cells, a study by Keris-Sen et al. demonstrated that sonication at 0.4 kWh/L significantly enhanced cell disruption [17]. This process nearly doubled lipid extraction efficiency compared to using solvents alone. However, more than 30% lipid remained in the biomass. The effects of different types of ultrasound apparatuses (cup horn, immersion horn, and cavitating tube) on the ultrasound-assisted solvent extraction of soybean-germ oil were evaluated [86]. The study found that the highest oil yield was achieved using a cavitating tube at 19 kHz, combined with double sonication using an additional immersion horn at 25 kHz. This approach improved oil yields by up to 500% compared to conventional methods and significantly enhanced extraction efficiency. The maximum extraction rate of 25.9% was achieved with a power of 65 W at 45 °C for 30 min, compared to a 4.8% extraction rate using conventional solvent extraction over 4 h. The study conducted by Martinez-Guerra et al. showed that ultrasound and microwave-assisted extraction enhanced extractive transesterification of algal lipids from Chlorella sp. using ethanol, improving yields over the conventional bench-top Bligh and Dyer method, which involves chloroform and methanol for isolating lipids [87]. They optimized the process conditions, finding that microwaves operating at 350 W for 5–6 min with a 1:12 algae to ethanol ratio achieved an 18.8% lipid yield and 96.2% fatty acid ethyl ester conversion. Ultrasonication at 490 W for 6 min with a 1:6 algae to ethanol ratio achieved a lipid extraction rate of 18.5% and fatty acid ethyl ester conversion rate of 95%, compared to the Bligh and Dyer method, which had a 13.9% yield and 78.1% conversion. In addition, ultrasound offered not only lipids extraction from algal Chlorella sp. but also transesterification in a short reaction time period. Both methods surpassed the Bligh and Dyer method in yield and conversion efficiency, indicating their potential for more efficient biodiesel production. Parniakov et al. examined ultrasound-assisted green solvent extraction of bioactive compounds from the microalgae Nannochloropsis spp. The study evaluated different solvents (water, ethanol, and dimethyl sulfoxide) for extracting phenolic compounds and chlorophylls [88]. When using individual solvents, the efficiency of recovery was highest with dimethyl sulfoxide, followed by ethanol and water. The yield of total phenolic compounds increased over time and reached saturation after 5 min, irrespective of the solvent employed. However, the extraction kinetics of total chlorophylls varied depending on the solvent used. In ethanol and water, the extraction yield plateaued after 7.5 min of ultrasound-assisted extraction. In contrast, in dimethyl sulfoxide, the yield peaked at 5 min and subsequently declined after 7.5 min. The decline in yield could be attributed to the increase in sample temperature due to prolonged ultrasound energy, leading to heat degradation of the chlorophyll.

Besides ultrasonication, pulsed electric field technology has also been integrated with solvent extraction to enhance intracellular compound extraction. A study demonstrated pulsed electric field pretreatment to enhance lipid recovery from the microalga Scenedesmus [89]. The study found that combining pulsed electric field with solvent extraction markedly increased the yield of crude lipids and fatty acid methyl esters compared to direct solvent extraction of untreated samples. Pulsed electric field pretreated biomass, when extracted with solvents, yielded up to 3.1 times more fatty acid methyl esters than untreated biomass and required fewer solvents than using solvent extraction from un-pretreated biomass. Under the optimal combination of pulsed electric field pretreatment and solvent extraction, a 12-fold reduction in solvent use while maintaining high lipid recovery efficiency was achieved. This suggests that pulsed electric field pretreatment is an effective method for enhancing lipid extraction from microalgae and reducing solvent consumption. Parniakov et al. investigated the pulsed electric field-assisted solvent extraction of valuable compounds from Nannochloropsis sp. using binary mixtures of organic solvents (dimethyl sulfoxide and ethanol) and water [90]. They compared a one-stage solvent extraction to a two-stage process involving initial pulsed electric field treatment followed by solvent extraction. The study found that pulsed electric field pretreatment significantly improved extraction efficiency, particularly for chlorophylls and carotenoids, and reduced the required solvent concentration. The two-stage process was more effective at extracting pigments and non-degraded proteins while minimizing solvent use. This approach demonstrated the potential of pulsed electric field to enhance extraction processes in a more sustainable manner by reducing solvent consumption and improving yield. Pulsed electric field technology has been proven effective as a pretreatment for enhancing lipid extraction from the green microalga Ankistrodesmus falcatus using the green solvent ethyl acetate [91]. The study showed that pulsed electric field pretreatment improved lipid recovery efficiency significantly compared to untreated samples. Pulsed electric field disrupted up to 90% of the cells, thereby reducing the required solvent contact time and accelerating lipid recovery. As a result, lipid yield increased to 6100 µg/L with pulsed electric field pretreatment, compared to 2600 µg/L without pretreatment. Pulsed electric fields were also used as pretreatment to assist solid–liquid extraction of aromatic and bioactive compounds from various plant sources including orange peels, vanilla pods, and cocoa bean shells [92]. The results demonstrated that pulsed electric field pretreatment with intensity of 3–5 kV cm⁻¹ and energy input as low as 15–40 kJ/kg enhanced the extraction yields significantly, demonstrating that pulsed electric field enhanced tissue permeabilization for extraction ability without degrading phenolic compounds.

Microwave-assisted extraction is an emerging technique for extracting lipids from wet microalgae, providing several advantages over traditional methods. Operating at a frequency of about 2.45 GHz, the microwave utilizes dielectric heating by absorbing energy in polar compounds present in the wet sample. This process causes polar molecules within the wet biomass to vibrate, increasing the temperature of the intracellular liquid. The resulting evaporation generates pressure on the cell walls, leading to their rupture and the release of lipids [93].

A number of studies have shown that microwave-assisted solvent extraction can enhance lipid yield and reduce extraction time from microalgae [94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109]. Cheng et al. developed a chloroform-free process for lipid extraction from wet microalgae Chlorella pyrenoidosa, employing microwave technology followed by hexane extraction [110]. The study identified 90 °C as the optimal temperature for this microwave-assisted solvent extraction process. This method efficiently converts triglycerides in wet microalgae into methyl esters, which are lower in molecular weight and, thus, dissolve more readily in hexane. As a result, the process enhanced the fatty acid methyl ester content in crude biodiesel to 86.7%. in addition, undesired polar pigments present in microalgae are not extracted during this process. Furthermore, pulsed microwave technology is considered an energy-saving pretreatment for microalgae. In a study by Zhang et al. using fresh Auxenochlorella protothecoides, the effects of pulse duration, repetition rate, and pulse power on cell permeabilization and lipid yield were assessed [111]. The results showed that pulsed microwaves at 2.8 kW peak power with 200 µs pulses significantly enhanced lipid yield up to 90% of the total lipid content through solvent extraction. Efficiency was primarily influenced by the total energy input rather than specific pulse properties, demonstrating a positive correlation between energy input and lipid yield. Additionally, pulsed microwaves consumed less energy (2.53 MJ/kg dry weight) compared to traditional hexane extraction methods, highlighting their potential for energy-efficient and scalable microalgae processing.

3.2. Combination of Physical and Biological Methods

Combining physical and biological extraction methods using enzymes to extract intracellular compounds provides an efficient technique to increase the yield and purity of targeted compounds. Physical methods like ultrasonication physically disrupt cell structures, enhancing the accessibility of enzymes to the intracellular components. Subsequently, specific enzymes can be employed to selectively break down complex molecules.

Integrating a physical method with enzyme digestion has shown effective extraction of protein from Chlorella species with a 72.4% extraction efficiency [41]. The study combined ethanol soaking, enzymatic digestion, ultrasonication, and homogenization techniques. This multifaceted approach significantly improved protein yield compared to traditional solvent extraction methods, providing a reference for efficient bioactive compound extraction from microalgal cells. Ultrasonic assisted enzymatic hydrolysis was also employed on the extraction of protein from excess sludge. Ultrasonic pretreatment combined with enzymatic hydrolysis significantly enhances protein extraction rates and improves sludge dewatering performance [112]. By optimizing conditions such as ultrasonic power density, hydrolysis time, enzyme dose, and pH, protein extraction rates of 55.9% and 52.3% for two types of sludge were achieved. Ultrasound-assisted enzyme extraction was also applied to extract carotenoids from yeast from a fermentation broth [113]. Another study demonstrated that ultrasound-assisted aqueous enzymatic extraction could be used to extract oil from gardenia fruits [114]. Two enzymes, Cellic CTec3 and Alcalase 2.4 L, were used with ultrasonic pretreatment. The optimal conditions achieved up to 18.7% extraction efficiency. The research discussed ultrasonic pretreatment led to more efficient cell wall disruption and higher oil yields. The physicochemical properties of the extracted oil were better using ultrasound-assisted aqueous enzymatic extraction, indicated by the lowest acid value and peroxide value. Further expanding on techniques, a study by Hu et al. investigated the extraction of oil from cherry seeds using an ultrasonic microwave-assisted aqueous enzymatic extraction process, achieving an oil recovery of 83.9%. The oil extracted under these conditions showed no significant difference in fatty acid compositions when compared to traditional methods but displayed better physicochemical properties and a higher content of bioactive constituents [115].

Table 5. Summary of studies on physical assisting techniques extracting intracellular compound.

Microorganism	Target Compounds	Key Finding	Source
Nannochloropsis sp., P. tricornutum and P. kessleri	Proteins and chlorophyll a	High-voltage electrical discharge-assisted solvent extraction was most effective for extracting carbohydrates, while ultrasonication-assisted solvent extraction was more efficient for extracting proteins and chlorophyll a from microalgae.	[83]
Fungi	Carotenoids	Ultrasound with sodium bicarbonate doubled the number of recovered carotenoids.	[84]
Foxtail millet bran	Phenolic compounds	Ultrasonic-assisted deep eutectic solvent extraction method produced higher total phenolics, total flavonoids, in vitro antioxidant activity, and acetylcholinesterase inhibitory activity than the conventional solvent extraction.	[85]
Wet microalgae	Lipid	Coupling sonication significantly enhanced cell disruption and doubled lipid extraction efficiency compared to using solvents alone.	[17]
Soybean germ	Lipids	The highest oil yield was achieved by ultrasound-assisted extraction using a cavitating tube at 19 kHz, combined with double sonication using an additional immersion horn at 25 kHz.	[86]
Chlorella sp.	Lipids	Ultrasound not only improved yields over conventional bench-top Bligh and Dyer method but also shortened the transesterification of algal lipids.	[87]
Nannochloropsis sp.	Phenolic compounds	Ultrasound extraction doubled the yield of total phenolic compounds compared to extraction without ultrasonication.	[88]
Scenedesmus	Lipids	Under the optimal combination of pulsed electric field pretreatment and solvent extraction, solvent use was reduced 12-fold while maintaining high lipid recovery efficiency.	[89]
Nannochloropsis sp.	Chlorophylls and carotenoids	Pulsed electric field pretreatment significantly improved extraction efficiency, particularly for chlorophylls and carotenoids, and reduced the required solvent concentration.	[90]
Ankistrodesmus falcatus	Lipids	Pulsed electric field pretreatment improved lipid recovery efficiency significantly compared to untreated samples extracted with green solvent ethyl acetate.	[91]
Plant	Aromatic and bioactive compounds	Pulsed electric field pretreatment with intensity of 3–5 kV cm−1 and energy input as low as 15–40 kJ/kg enhanced the solid–liquid extraction yields significantly.	[92]
Chlorella pyrenoidosa	Lipids	Microwave-assisted hexane extraction can replace the traditional chloroform method.	[110]
Auxenochlorella protothecoides	Lipids	Pulsed microwaves consume less energy compared to traditional extraction methods.	[111]
microalgae Chlorella	Protein	A multifaceted approach combining ethanol soaking, enzymatic digestion, ultrasonication, and homogenization techniques significantly improved protein yield compared to traditional methods.	[41]
Excess sludge	Protein	Ultrasonic pretreatment combined with enzymatic hydrolysis significantly enhances protein extraction rates and improves sludge dewatering performance.	[112]
Gardenia fruits	Oil	Physicochemical properties of the extracted oil were better using ultrasound-assisted aqueous enzymatic extraction, indicated by the lowest acid value and peroxide value.	[114]
Cherry seeds	Oil	An ultrasonic microwave-assisted aqueous enzymatic extraction process displayed better physicochemical properties and a higher content of bioactive constituents.	[115]

Table 5. Summary of studies on physical assisting techniques extracting intracellular compound.

Microorganism	Target Compounds	Key Finding	Source
Nannochloropsis sp., P. tricornutum and P. kessleri	Proteins and chlorophyll a	High-voltage electrical discharge-assisted solvent extraction was most effective for extracting carbohydrates, while ultrasonication-assisted solvent extraction was more efficient for extracting proteins and chlorophyll a from microalgae.	[83]
Fungi	Carotenoids	Ultrasound with sodium bicarbonate doubled the number of recovered carotenoids.	[84]
Foxtail millet bran	Phenolic compounds	Ultrasonic-assisted deep eutectic solvent extraction method produced higher total phenolics, total flavonoids, in vitro antioxidant activity, and acetylcholinesterase inhibitory activity than the conventional solvent extraction.	[85]
Wet microalgae	Lipid	Coupling sonication significantly enhanced cell disruption and doubled lipid extraction efficiency compared to using solvents alone.	[17]
Soybean germ	Lipids	The highest oil yield was achieved by ultrasound-assisted extraction using a cavitating tube at 19 kHz, combined with double sonication using an additional immersion horn at 25 kHz.	[86]
Chlorella sp.	Lipids	Ultrasound not only improved yields over conventional bench-top Bligh and Dyer method but also shortened the transesterification of algal lipids.	[87]
Nannochloropsis sp.	Phenolic compounds	Ultrasound extraction doubled the yield of total phenolic compounds compared to extraction without ultrasonication.	[88]
Scenedesmus	Lipids	Under the optimal combination of pulsed electric field pretreatment and solvent extraction, solvent use was reduced 12-fold while maintaining high lipid recovery efficiency.	[89]
Nannochloropsis sp.	Chlorophylls and carotenoids	Pulsed electric field pretreatment significantly improved extraction efficiency, particularly for chlorophylls and carotenoids, and reduced the required solvent concentration.	[90]
Ankistrodesmus falcatus	Lipids	Pulsed electric field pretreatment improved lipid recovery efficiency significantly compared to untreated samples extracted with green solvent ethyl acetate.	[91]
Plant	Aromatic and bioactive compounds	Pulsed electric field pretreatment with intensity of 3–5 kV cm−1 and energy input as low as 15–40 kJ/kg enhanced the solid–liquid extraction yields significantly.	[92]
Chlorella pyrenoidosa	Lipids	Microwave-assisted hexane extraction can replace the traditional chloroform method.	[110]
Auxenochlorella protothecoides	Lipids	Pulsed microwaves consume less energy compared to traditional extraction methods.	[111]
microalgae Chlorella	Protein	A multifaceted approach combining ethanol soaking, enzymatic digestion, ultrasonication, and homogenization techniques significantly improved protein yield compared to traditional methods.	[41]
Excess sludge	Protein	Ultrasonic pretreatment combined with enzymatic hydrolysis significantly enhances protein extraction rates and improves sludge dewatering performance.	[112]
Gardenia fruits	Oil	Physicochemical properties of the extracted oil were better using ultrasound-assisted aqueous enzymatic extraction, indicated by the lowest acid value and peroxide value.	[114]
Cherry seeds	Oil	An ultrasonic microwave-assisted aqueous enzymatic extraction process displayed better physicochemical properties and a higher content of bioactive constituents.	[115]

4. Challenges and Future Perspectives

While physical disruption techniques have shown considerable promise, they come with their own set of limitations of challenges. One of the key limitations of physical methods is their lack of selectivity compared to the well-established chemical methods. Chemical methods, particularly solvent-based extractions, offer great selectivity by using tailored solvent to selectively extract targeted compounds without dissolving unwanted cellular contents, resulting in high-purity final products, and simplifying the downstream purification process. In contrast, physical methods, like HPH and mechanical milling, rely on mechanical forces to indiscriminately disrupt the cells, leading to the non-specific release of all intracellular components, including cell debris and other unwanted intracellular organelles. Thus, the extracted compounds by using physical methods usually have a lower purity compared to those extracted by using solvent extraction, thereby complexing the downstream process as additional filtration or clarification are often needed to remove those unwanted byproducts. Although advanced techniques, such as ultrasound and pulsed electric field, offer relatively higher selectivity to HPH and mechanical milling, they still fall short in product purity and selectivity compared to chemical methods.

Second, while physical methods avoid the use of toxic and environmentally unsustainable organic solvents, they often come at the expense of higher energy consumption, particularly of electrical energy [116]. Mechanical milling, for instance, consumes intensive electricity to constantly agitation beads and rotate large drums, and both HPH and ultrasounds also need significant electricity to maintain the high pressure or ultrasonic waves in liquid medium for cell disruption. When developing these technologies at a lab-scale, the energy consumption is often overlooked due to the small-scale operation; however, the energy requirement is enormous in continuous industrial processes and can significantly increase operating costs. Therefore, the economics and environmental aspects of the commercialization of these methods should be considered at the early stages.

Third, some physical methods, such as mechanical milling and HPH, can generate large amounts of heat as a byproduct of the mechanical forces applied to break the cells walls. The heat can increase the liquid-medium temperature and potentially cause thermal damage to heat-sensitive bioactive compounds, such as therapeutic proteins, enzymes, and certain bioactive compounds [117]. Although some cooling systems, such as water cooling and air cooling, can be integrated into these physical disruption methods, it increases the equipment cost and operating complexity of the system.

To address these challenges, further research and development are needed to improve the efficiency, selectivity, and sustainability of physical cell disruption methods for intracellular compound recovery. Although most physical methods, like HPH and high-pressure homogenization, experience low-selectivity challenges, techniques such as pulsed electric field and ultrasound offer comparatively better selectivity among physical methods, making them promising candidates for further development. Currently, while these two techniques still do not match the precision level of organic solvent extraction, we can precisely optimize the key processing parameters, such as ultrasound frequency, duration, electric field strength, medium conductivity, to specific cell types and intracellular compounds for increasing the selectivity and purity of targeted compounds. In addition, beyond individual physical methods, combining physical and chemical approaches could significantly improve selectivity. For instance, the integration of membrane filtration systems with physical methods could facilitate the simultaneous removal of cell debris and impurities during the disruption process.

Reducing energy consumption of the physical methods is critical for improving the technology adoption and economics. In addition to the traditional ways to reduce energy through the improvements of equipment design (e.g., bead size in mechanical milling) and optimization of operating conditions (e.g., HPH pressure, electric field strength), the latest machine learning techniques, especially the new concept of the digital twin system, offer grand opportunities to control the entire processes and optimize energy use. A digital twin is a virtual model of a physical system that can be used to simulate the entire bioprocess, allowing real-time data to be analyzed and used for predictive process control. By integrating digital twins with machine learning algorithms, engineers can dynamically optimize parameters like pressure, pulse frequency, and power consumption, ensuring that energy use is minimized while enhancing the product quality and process efficiency.

Lastly, it is important to incorporate techno-economic analysis (TEA) and life cycle assessment (LCA) into the early stage of the development of the methods, to endure that newly developed physical disruption methods are both economically viable and environmentally sustainable. By integrating TEA and LCA, engineers can not only assess the overall economic and environmental metrics of a process, but also identify the bottlenecks regarding the cost and energy consumption before the technology reaches the pilot or commercial stage. This allows for more informed decision making to select appropriate technical routes, such as whether to increase the HPH pressure to enhance product recovery or maintain the pressure to reduce the capital and operating (energy) cost to make the whole process economically feasible and environmentally sustainable.

5. Conclusions

In summary, the use of physical disruption techniques marks a crucial step forward in extracting intracellular compounds, potentially offering a more environmentally sustainable and effective option compared to traditional chemical and biological methods. Through the application of high-pressure homogenization, ultrasonication, milling, and pulsed electric fields, high yields of intracellular compounds can be achieved with fewer negative impacts on the environment. These methods not only address the environmental concerns associated with conventional chemical extraction but also simplify the processing steps. Physical disruption technology has great potential in intracellular compound extraction. As bioprocessing continues to be focused on sustainability, these physical methods will gain more applications. Continued improvements in the equipment and processing parameters, as well as developments of novel physical destruction techniques, are expected to unlock possibilities for physical extracting valuable compounds independently from a variety of biological sources. Looking forward, the significance of physical disruption techniques in extracting intracellular compounds is expected to increase, driven by the need for cleaner, more efficient, and more environmentally friendly processes. Continued research and innovation in this field are likely to broaden our understanding and enhance our bioprocessing capabilities.