Cell Disruption Technologies Research Articles

Physical Cell Disruption Technologies for Intracellular Compound Extraction from Microorganisms

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.

Enhancing protein extraction from heterotrophic Auxenochlorella protothecoides microalgae through emerging cell disruption technologies combined with incubation

This study evaluated pulsed electric fields (PEF) and ultrasonication (US) combined with incubation to enhance cell disruption and protein extraction from Auxenochlorella protothecoides, comparing them to conventional high-pressure homogenization (HPH). A 5 h incubation enhanced protein yield by 79.4 % for PEF- and 27.2 % for US-treated samples. Extending the incubation to 24 h resulted in a total yield increase of 122 % for PEF (0.25 ± 0.03 kgEP kgTP−1) and 51.9 % for US (0.20 ± 0.02 kgEP−1 kgTP−1). Autofermentation in untreated cells after 24 h resulted in protein release with lower yields than all other treated and incubated samples. While HPH had the highest protein yield (0.58 ± 0.04 kgEP kgTP−1), PEF-incubation after 5 h (56.6 ± 5.3 MJ kgEP−1) and 24 h (49.5 ± 3.7 MJ kgEP−1) were 1.5 and 1.7-times more energy-efficient than HPH (82.9 ± 7.8 MJ kgEP−1). PEF combined incubation is an energy-efficient and targeted protein extraction method in heterotrophic A. protothecoides biorefinery.

Evaluation of cell disruption technologies on magnetosome chain length and aggregation behaviour from Magnetospirillum gryphiswaldense MSR-1.

Magnetosomes are biologically-derived magnetic nanoparticles (MNPs) naturally produced by magnetotactic bacteria (MTB). Due to their distinctive characteristics, such as narrow size distribution and high biocompatibility, magnetosomes represent an attractive alternative to existing commercially-available chemically-synthesized MNPs. However, to extract magnetosomes from the bacteria, a cell disruption step is required. In this study, a systematic comparison between three disruption techniques (enzymatic treatment, probe sonication and high-pressure homogenization) was carried out to study their effect on the chain length, integrity and aggregation state of magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 cells. Experimental results revealed that all three methodologies show high cell disruption yields (>89%). Transmission electron microscopy (TEM), dynamic light scattering (DLS) and, for the first time, nano-flow cytometry (nFCM) were employed to characterize magnetosome preparations after purification. TEM and DLS showed that high-pressure homogenization resulted in optimal conservation of chain integrity, whereas enzymatic treatment caused higher chain cleavage. The data obtained suggest that nFCM is best suited to characterize single membrane-wrapped magnetosomes, which can be particularly useful for applications that require the use of individual magnetosomes. Magnetosomes were also successfully labelled (>90%) with the fluorescent CellMask™ Deep Red membrane stain and analysed by nFCM, demonstrating the promising capacity of this technique as a rapid analytical tool for magnetosome quality assurance. The results of this work contribute to the future development of a robust magnetosome production platform.

Extraction of Valuable Biomolecules from the Microalga Haematococcus pluvialis Assisted by Electrotechnologies.

The freshwater microalga Haematococcus pluvialis is well known as the cell factory for natural astaxanthin, which composes up to 4-7% of its total dry weight. The bioaccumulation of astaxanthin in H. pluvialis cysts seems to be a very complex process that depends on different stress conditions during its cultivation. The red cysts of H. pluvialis develop thick and rigid cell walls under stress growing conditions. Thus, the biomolecule extraction requires general cell disruption technologies to reach a high recovery rate. This short review provides an analysis of the different steps in H. pluvialis's up and downstream processing including cultivation and harvesting of biomass, cell disruption, extraction and purification techniques. Useful information on the structure of H. pluvialis's cells, biomolecular composition and properties and the bioactivity of astaxanthin is collected. Special emphasis is given to the recent progress in application of different electrotechnologies during the growth stages and for assistance of the recovery of different biomolecules from H. pluvialis.

Mechanical Cell Disruption Technologies for the Extraction of Dyes and Pigments from Microorganisms: A Review

The production of pigments using single cell microorganisms is gaining traction as a sustainable alternative to conventional syntheses, which rely, in no negligible proportions, on petrochemicals. In addition to depending on petroleum, these syntheses involved the use of toxic organic solvents, which may be inadequately disposed of across a range of industries, thus compounding the deleterious effects of fossil fuel exploitation. Literature suggests that notable research efforts in the area of sustainable pigment production using single cell microorganisms are focused on the production of pigments coveted for their interesting qualities, which transcend their mere capacity to dye various fabrics both natural and synthetic. As interest in sustainable pigment biosynthesis grows, the need to devise effective and efficient cell disruption processes becomes more pressing given that the viability of pigment biosynthesis is not only dependent on microorganisms’ yield in terms of production, but also on researchers’ ability to recover them. This review chiefly reports findings as to mechanical cell disruption methods, used individually or in various combinations, and their aptitude to recover biosynthetic pigments.

Strategies for increasing lipid accumulation and recovery fromY. lipolytica: A review

Microbial-based biodiesel is produced by transesterification of lipids extracted from microbial cells, and is considered as a potential replacement of fossil fuel due to its advantages in reducing greenhouse gas emissions.Yarrowia lipolyticais one of the most studied oleaginous yeasts able to produce lipids under some fermentation conditions and is considered as a potential industrial host for biodiesel production. Several approaches have been evaluated to increase the economical attraction of biodiesel production fromY. lipolyticalipids. In this review, we highlighted the different strategies reported in the literature, allowing this yeast to achieve high lipid accumulation. These include metabolic engineering strategies, the use of low-cost effective substrates, and the optimization of the cultivation conditions for higher lipid productivity and less operating cost. We also summarized the most effective cell disruption technologies that improve the extraction efficiencies of lipids fromY. lipolytica.

Hypotonic osmotic shock treatment to enhance lipid and protein recoveries from concentrated saltwater Nannochloropsis slurries

This study reports the use of hypotonic osmotic shock as a treatment step to enhance the recoveries of biofuel-convertible lipids and proteins from lipid-rich saltwater Nannochloropsis gaditana (N. gaditana) slurries (biomass content = ~140 mg biomass / g slurry, total lipid content = ~600 mg lipid /g biomass). The osmotic shock was induced through repeated washing of microalgal slurries with multiple batches of fresh water. Subjecting the slurries to 2 stages of freshwater washing resulted in a measurable damage to cell membranes (the uptake of membrane permeability marker increased by 6 folds), a partial loss of cell viability (only 64% of available cells were recoverable), and a minor release of free protein (~2 wt% of available protein) from the biomass into the interstitial space of the slurries. Hypotonic osmotic shock was revealed to be ineffective in rupturing N. gaditana slurries (only 13 ± 9% of available cells were ruptured after 2-stage washing) and, as such, had a limited prospect as a stand-alone cell disruption technology for the saltwater strain.The washing treatment, however, was found to be able to weaken the structural integrity of N. gaditana slurries and enhance the performance of subsequent mechanical or chemical cell disruption technologies when installed as a preparatory step. Applying the washing treatment prior to high-pressure homogenisation (HPH) and low-solvent-to-biomass ratio hexane extraction (hexane : slurry = 1:1 w/w) for the recovery of biofuel-convertible lipids increased the extent of cell rupture from 28 ± 8 to 46 ± 19% of available cells and more than doubled neutral lipid yield from 25.1 ± 2.0 to 64.6 ± 4.9 wt% of available neutral lipid. Initial analysis revealed that the washing treatment had a minimal energy cost (~6% of the total energy expenditure of downstream processing) and that its integration into HPH + hexane lipid recovery led to a 2.5 fold increase in the energy output of the biomass. Partnering the washing treatment with NaOH hydrolysis increased protein yield from 6.7 ± 2.4 to 31.9 ± 10.7 wt% of available protein.

The construction of a green and efficient system for the separation of polyprenols from Ginkgo biloba leaves

The aim of this work was to construct a new type of system for the green extraction and efficient separation of Ginkgo biloba leaf polyprenols (GBP). First, cell disruption technologies (enzymolysis, ultrasound, microwave and ball milling) coupled with deep eutectic solvents (DES) were used to extract polyprenyl acetates (PA) from Ginkgo biloba leaves. Results showed that ball milling was the best assisted extraction method, and the amount of PA was 89.88 mg/g. Then, a novel silver-loaded material with selectivity for PA was synthesized, characterized and utilized to absorb PA from the DES extraction solution. The purity of the PA quickly reached 71.28 % after being separated by the silver-loaded material. Finally, saponification and molecular distillation were adopted to remove non-GBP ingredients, and a high-purity GBP (99.80 %) was obtained by optimizing its processing conditions. This study provides a technical support for the development of a green and efficient GBP separation method and lays the foundation for the fabrication of GBP-based drugs, health products and cosmetics.

Combination of cell disruption technologies for lipid recovery from dry and wet biomass of Yarrowia lipolytica and using green solvents

This work aims to investigate the efficiency of bead milling combined or not with high-pressure homogenization on the cell disruption and lipid recovery from Yarrowia lipolytica oleaginous yeast. First, a simulation study involving the use of the Hansen solubility parameters’ approach was performed in order to identify, among 41 conventional and “green” solvents, the most promising ones that are able to replace n-hexane for lipid recovery from Y. lipolytica biomass. The results obtained showed that the pre-treatment involving both high-pressure homogenization and bead milling applied sequentially was more performant than that involving bead milling alone. In addition, bead-milling parameters were optimized showing an optimal bead size of 4.9 mm and a processing time of 30 s. Among the tested solvents, isoamyl acetate was selected as the most appropriate “green” solvent, maximizing the lipid extraction, compared to n-hexane. Despite the better performance of the dry route compared to the wet one, promising results were obtained towards 1) minimizing the energy consumed and 2) replacing n-hexane by “green” solvents for lipid recovery from Y. lipolytica yeast.

Effect of pulsed electric fields and high pressure homogenization on the aqueous extraction of intracellular compounds from the microalgae Chlorella vulgaris

Pulsed Electric Fields (PEF) and High Pressure Homogenization (HPH) are promising and scalable cell disruption technologies of microalgae cells. In this work, the permeabilization degree, morphological properties, and extractability of intracellular compounds from microalgae Chlorella vulgaris suspensions (1.2%, w/w) were investigated as a function of PEF treatment at different electric field strengths (10–30 kV/cm) and total specific energy input (20–100 kJ/kg), in comparison with the more disruptive HPH treatment (150 MPa) at different number of passes (nP = 1–10). The conductivity and the particle size analyses, as well as the SEM images, clearly showed that PEF induces the permeabilization of the cell membranes in an intensity-dependent manner, without producing any cell debris, whereas HPH treatment causes the total disruption of the algae cells into small fragments. Coherently with the lower permeabilization capability, PEF promoted the selective extraction of carbohydrates (36%, w/w, of total carbohydrates), and low molecular weight proteins (5.2%, w/w, of total proteins). On the other hand, HPH induced the undifferentiated release of all the intracellular content, resulting in a 1.1 and 10.3 fold higher yields than PEF, respectively of carbohydrates and protein.These results suggest that, in a multi-stage biorefinery, PEF could represent a suitable cell disruption method for the selective recovery of small-sized cytoplasmic compounds, while HPH should be placed at the end the cascade of operations allowing the recovery of high molecular weight intracellular components.

Evaluating the potential of cell disruption technologies for green selective extraction of antioxidant compounds from Stevia rebaudiana Bertoni leaves

An increased interest has been shown by both food technologists and food industry regarding Stevia rebaudiana Bertoni leaves (Stevia) for their high content of bioactive components (phenolic compounds, vitamin C, carotenoids). The aim of this work was to study the effect of emerging technologies such as high voltage electrical discharges (HVED) and pulsed electric fields (PEF) and ultrasounds (US) on the intensification of the extraction of valuable compounds from Stevia leaves. The proposed processes combined pretreatment (HVED, PEF and US) and extraction of intracellular compounds using water as solvent at ambient temperature. The energy inputs of the treatments varied from 24 to 141kJ/kg and the results were compared to control diffusion experiments. Chlorophyll a, and b, total carotenoid, total phenolic compounds and antioxidant capacity (DPPH) were determined. A significant increase in soluble matter and antioxidant compounds was found after HVED, PEF and US-assisted extraction as compared to control samples. Results showed that HVED, PEF and US treatments improved both kinetics and extraction yield of soluble matter. These results show the ability of HVED to be used as a potential technology to enhance protein recovery using water and avoiding the use of other solvents and grinding. Chlorophyll content was significantly higher (3-fold increase) after HVED assisted extraction at 141kJ/kg in comparison to control sample (0.352 and 0.355mg/L for chlorophyll a and b, respectively). The same energy input for HVED permitted attain highest extraction diffusivities of total soluble matter (D=3.06×10−09m2/s), followed by total phenolic compounds (D=2.60×10−10m2/s) and then proteins (D=6.00×10−11m2/s).

New Approaches for the Use of Non-conventional Cell Disruption Technologies to Extract Potential Food Additives and Nutraceuticals from Microalgae

In recent decades, microalgae species have focused the attention of several research groups and food industry as they are a great source of nutritionally valuable compounds. The use of environmentally friendly technologies has led to researchers and food industry to develop new alternative processes that can extract nutritionally valuable compounds from different sources, including microalgae. This note describes the potential use of some non-conventional methods including sub- and supercritical fluid extraction, pulsed electric fields, high-voltage electric discharges, high-pressure homogenization, ultrasound- and microwave-assisted extraction, which involve cell disruption to recover nutritionally valuable compounds from microalgae and can help to comply with criteria of green chemistry concepts and sustainability.

Optimizing recombinant product recovery through improvements in cell-disruption technologies

Although several variations on traditional disruption equipment have been developed over the past five years, no radical changes have occurred in cell-disruption technologies. Major efforts have, however, been put into the containment features of disruption equipment to ensure safe operation with recombinant organisms. Combinations of technologies, especially chemical pre-treatment followed by mechanical disruption, will improve disruption efficiency. If existing equipment can be used with enhanced productivity, then it is a bonus in good manufacturing practice operations because operating procedures and protocols merely have to be amended. A lack of understanding of, and/or information on, production-scale techniques, as well as the limitations of the equipment available, has often hindered the development of potential products. As more and more recombinant-based processes emerge, it is essential that bench-scale research is carried out using equipment that will scale up effectively and safely.