Physical Disruption Of Cells 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 phycocyanin yield from Spirulina sp. under salt stress using various extraction methods.

Phycocyanin, a blue-coloured pigment, predominantly found and derived from Spirulina sp., has gained researchers' interest due to its vibrant hues and other attractive properties like antioxidant and anti-microbial. However, the lack of reliable and sustainable phycocyanin extraction strategies without compromising the quality has hindered the scaling up of its production processes for commercial purposes. Here in this study, phycocyanin was extracted from wet and dry biomass Spirulina sp., using three different physical cell disruption methods (ultrasonication, homogenization, and freeze-thaw cycles) combined with two different buffers (phosphate buffer and acetate buffer) and water (as control). The result showed that the freeze-thaw method combined with acetate buffer produced the highest yield (25.013 ± 2.572mg/100mg) with a purity ratio of 0.806 ± 0.079. Furthermore, when subjected to 30% w/v salt stress, 1.9 times higher phycocyanin yield with a purity ratio of 1.402 ± 0.609 was achieved using the previously optimized extraction method.

Energy assessment and enhancement of the lipid yield of indigenous Chlorella sp. KA-24NITD using Taguchi approach

Abstract A major step in microalgal biodiesel production is lipid extraction, which is mostly done using the conventional chloroform-methanol solvent system. The objective of the present work was to optimise the process of pretreatment of an indigenous Chlorella sp. KA-24NITD (Chl-24). Various physical cell disruption methods were combined with the optimised solvent system along with chemical (Dimethyl sulfoxide) and enzymatic (cellulase and pectinase) pretreatments of wet and dry biomass for lipid yield enhancement. Optimisation was done by Taguchi Orthogonal Array L9 (34). Under optimised conditions, subsequent experimental runs showed that the optimum lipid yield of about 38.59% was obtained when wet biomass was enzymatically pretreated with pectinase and cellulase in the ratio 1:2 along with the combination of the physical process. Thus enzymatic pretreatment proved to be more efficient than chemical pretreatment showing significant results by improving lipid yield, which suggests that Chl-24 could be a potent candidate for biodiesel production.

Processing of astaxanthin-rich Haematococcus cells for dietary inclusion and optimal pigmentation in Rainbow trout, Oncorhynchus mykiss L.

A range of physical cell disruption techniques have been evaluated to aid the processing of astaxanthin-rich haematocysts of Haematococcus pluvialis for inclusion in salmonid feeds. Cell disruption by a scalable pressure treatment system was shown to be effective in breaking open the haematocysts without altering the content or isomeric composition of carotenoids in the algal cells. Storage of disrupted cells was optimal at −20°C in the dark under nitrogen. Disrupted cells were spray-dried, incorporated into commercial diets and fed to rainbow trout (Oncorhynchus mykiss L.). A marketable level of pigmentation in fish muscle was achieved after 10-week dietary supplementation. The geometric and optimal isomer composition of the astaxanthin deposited in the muscle was nearly identical to that seen in Haematococcus. Changes were observed in the chirality of the astaxanthin deposited in the skin in comparison with that isolated from both the white muscle and the alga.

Evaluation of different RNA extraction methods of filamentous fungi in various food matrices

Aflatoxin producing-moulds may grow on different foods (e.g. cereals, ripened meats) and contaminate such products with aflatoxins. Molecular studies about expression of genes involved in these secondary metabolites biosynthesis require isolation of good quality mould RNA from foods. In this work, six different RNA extraction methods, which combine physical cell disruption protocols and RNA purification techniques, were evaluated in different mouldy foods to obtain intact mould RNA and without these food inhibitors. For this, five food matrices (dry-cured ham, tea, paprika, peanut and wheat) were inoculated with Aspergillus parasiticus. Protocols that used bead beating as physical cell disruption and TRIzol yielded RNA of low quality. Differences in RNA yield were found between the two tested commercial kits. The ‘mortar-pestle RNeasy’ protocol which consists of freezing the mycelium with liquid nitrogen and grinding it with the aid of a mortar and pestle and the RNeasy kit could be utilised to obtain intact RNA from a wide range of aflatoxin-producing fungi of good quantity and quality from contaminated foods.

The contribution of apoptosis and necrosis in freezing injury of sea urchin embryonic cells

Sea urchins have recently been reported to be a promising tool for investigations of oxidative stress, UV light perturbations and senescence. However, few available data describe the pathway of cell death that occurs in sea urchin embryonic cells after cryopreservation. Our study is focused on the morphological and functional alterations that occur in cells of these animals during the induction of different cell death pathways in response to cold injury. To estimate the effect of cryopreservation on sea urchin cell cultures and identify the involved cell death pathways, we analyzed cell viability (via trypan blue exclusion test, MTT assay and DAPI staining), caspase activity (via flow cytometry and spectrophotometry), the level of apoptosis (via annexin V-FITC staining), and cell ultrastructure alterations (via transmission electron microscopy). Using general caspase detection, we found that the level of caspase activity was low in unfrozen control cells, whereas the number of apoptotic cells with activated caspases rose after freezing-thawing depending on cryoprotectants used, also as the number of dead cells and cells in a late apoptosis. The data using annexin V-binding assay revealed a very high apoptosis level in all tested samples, even in unfrozen cells (about 66%). Thus, annexin V assay appears to be unsuitable for sea urchin embryonic cells. Typical necrotic cells with damaged mitochondria were not detected after freezing in sea urchin cell cultures. Our results assume that physical cell disruption but not freezing-induced apoptosis or necrosis is the predominant reason of cell death in sea urchin cultures after freezing-thawing with any cryoprotectant combination.

Comparison of chemical washing and physical cell-disruption approaches to assess the surface adsorption and internalization of cadmium by Cupriavidus metallidurans CH34

Bacterial biosorption of heavy metals is often considered as a surface complexation process, without considering other retention compartments than cell walls. Although this approach gives a good description of the global biosorption process, it hardly permits the prediction of the fate of biosorbed metals in the environment. This study examines the subcellular distribution of cadmium (Cd) in the metal-tolerant bacterium Cupriavidus metallidurans CH34 through the comparison of an indirect chemical method (washing cells with EDTA) and a direct physical method (physical disruption of cells). The chemical washing approach presented strong experimental biases leading to the overestimation of washed amount of Cd, supposedly bound to cell membranes. On the contrary, the physical disruption approach gave reproducible and robust results of Cd subcellular distribution. Unexpectedly, these results showed that over 80% of passively biosorbed Cd is internalized in the cytoplasm. In disagreement with the common concept of surface complexation of metals onto bacteria the cell wall was poorly reactive to Cd. Our results indicate that metal sorption onto bacterial surfaces is only a first step in metal management by bacteria and open new perspectives on metal biosorption by bacteria in the environment, with implications for soil bioremediation or facilitated transport of metals by bacteria.

Role of Protein Disulfide Isomerase (PDI) In the Expression of Tissue Factor (TF) Procoagulant Activity on Apoptotic Myeloblasts

AbstractAbstract 152Disseminated intravascular coagulation (DIC) is a common and potentially life-threatening complication of acute myelogenous leukemia (AML). Although various components of the procoagulant and fibrinolytic systems have been implicated in its pathogenesis, aberrant expression of TF appears to be critically involved. Despite the need for more specific hemostatic agents, the molecular pathways underlying the expression of TF procoagulant activity (PCA) in AML remain obscure. In this study, we sought to identify determinants for systemic coagulation activation in a prospective cohort of 50 patients with newly diagnosed AML and conducted a series of in-vitro experiments to further characterize TF PCA regulation on myeloblasts in light of recently provided evidence. AML patients had significantly elevated plasma D-dimer and TF-specific PCA of isolated peripheral blood mononuclear cells (PB-MNCs) compared to healthy controls (n=10) and patients with ALL (n=5) or MDS (n=7), demonstrating specific upregulation of the TF-dependent coagulation pathway. In the AML cohort, TF PCA of PB-MNCs was 30-fold higher and TF+ plasma microparticles were more frequently detectable in patients with hypofibrinogenemia and a thrombohemorrhagic syndrome (n=8) as compared to patients with normal fibrinogen levels (n=42), providing further evidence that TF over-expression by myeloblasts is indeed a determinant for DIC decompensation. Physical cell disruption resulted in a 3-fold increase in TF PCA of PB-MNCs, indicating that a significant proportion of TF was non-coagulant (i.e. cryptic) on unperturbed cells. In patients with AML, TF PCA of PB-MNCs, absolute numbers of circulating blasts and lactate dehydrogenase (LDH) serum levels, a surrogate marker for leukemic cell turnover, were significantly correlated with plasma D-dimer, suggesting that membrane alterations associated with apoptosis/necrosis played a crucial role in myeloblast TF PCA expression. Extracellular PDI, which is abundantly expressed in the endoplasmic reticulum, has recently been implicated in TF regulation via its oxidoreductase and chaperone activity. In monoblastic THP1 cells harvested at varying time points during maintenance culture (n=5), we found a significant correlation between membrane PDI expression, as assessed by flow cytometry, and cell-associated TF PCA (r=0.93). Analysis of annexin V-FITC binding revealed that PDI was predominantly expressed on apoptotic monoblasts. Treatment of THP1 cells for 24 h with 1 μ M daunorubicin (DNR), a cytotoxic drug commonly used in AML therapy, induced >80% apoptosis and increased cellular TF PCA 8 ± 3-fold (n=8) independent of de-novo protein synthesis. Consistently, apoptotic cells stained extensively positive for PDI by flow cytometry. DNR-induced TF PCA was inhibited by 70 ± 6% (n=8) when THP1 cells were coincubated with RL90, an inhibitory PDI monoclonal antibody previously shown to diminish fibrin and platelet deposition in murine models of micro- and macrovascular thrombosis. Importantly, RL90 had no effect when added to THP1 cells at the end of the 24-h culture period, indicating specific effects on the expression of cellular TF PCA rather than the coagulation reaction itself. Similarly, coincubation of DNR-treated THP1 cells with the PDI inhibitor bacitracin dose-dependently (1-5 mM) inhibited TF activation by up to 93 ± 10% (n=3), while only a 30 ± 16% inhibition was observed when bacitracin was added at the end of the culture period. Virtually identical results were obtained with the AML cell lines, HL60 and U937. Furthermore, ex-vivo treatment of isolated myeloblasts from a patient with acute promyelocytic leukemia and decompensated DIC with 1 μ M DNR for 24 h induced >90% apoptosis and increased cellular TF PCA 5-fold. Extensive PDI staining was observed on apoptotic myeloblasts, and coincubation with bacitracin (5 mM) completely inhibited DNR-induced upregulation of TF PCA while not affecting baseline TF PCA expression. Finally, treatment with DNR resulted in the leakage of both LDH and PDI into the culture supernatants of AML cell lines, and soluble PDI antigen was detectable in the plasma samples of AML patients with the highest LDH serum levels. In summary, our findings suggest that, in addition to negatively charged phospholipids, surface-expressed and/or released PDI may be required for the efficient expression of TF-specific PCA on apoptotic myeloblasts. Disclosures:No relevant conflicts of interest to declare.

Amplification Facilitators and Pre-Processing Methods for PCR Detection of Strictly Anaerobic Beer-Spoilage Bacteria of the Class Clostridia in Brewery Samples

The aim of this study was to evaluate easy pre-PCR processing procedures to allow rapid and reliable detection of strictly anaerobic beer-spoilage bacteria throughout the brewing process by end-point and real-time PCR techniques. The efficiencies of the new procedures were evaluated using spiked brewery samples and specific PCRs for the target group bacteria. We found for the first time that the inclusion of 0.25% (w/v) bovine serum albumin (BSA) or 0.5% (w/v) polyvinyl pyrrolidone (PVP) in the end-point PCR mixture reduces the inhibiting effect of brewery sample extracts (3–10%, v/v) on PCR. Membrane filtration with a PVP or a sodium tri-polyphosphate-EDTA wash, and cross-flow filtration were the most promising new methods to reduce inhibitors from beer samples before cell lysis. Together with BSA, they allowed the analysis of 10% (v/v) of crude extracts instead of <3% (v/v). Moreover, we developed a one-hour procedure to prepare target DNA from process samples containing brewer's yeast. It involved removal of inhibitors by a two-step centrifugation followed by physical disruption of cells. The detection limit of the procedure was 101-103 CFU/mL. The developed procedures help to reduce the risk of partial or complete PCR failure due to inhibition and target DNA losses, with minimal sample handling.

Characterization and on-line monitoring of cell disruption and lysis using dielectric measurement

The dielectric measurement of Escherichia coli suspension was carried out between 0.01 and 100 MHz during cell disruption using a physical method or cell lysis induced by chemicals for the purpose of on-line monitoring of cell disruption or lysis of bacterial cells. The dielectric dispersion of relative permittivity centered at about 1.4 MHz and the dielectric parameters based on the fitting calculation well reflected the damage of these cells under physical and chemical stresses. The degree of cell disruption as determined by the dielectric parameters, the amplitude of dispersion and the conductivity, well corresponded to those obtained by other conventional kinetic analyses. This methodology can be utilized for the on-line monitoring of physical cell disruption and cell lysis induced by detergent and could provide direct information to control these processes. Based on these results, the dielectric measurement was successfully applied to monitor the stress-mediated cultivation of E. coli cells.

Cleavage of Nuclear DNA into Oligonucleosomal Fragments during Cell Death Induced by Fungal Infection or by Abiotic Treatments.

It is often claimed that programmed cell death (pcd) exists in plants and that a form of pcd known as the hypersensitive response is triggered as a defense mechanism by microbial pathogens. However, in contrast to animals, no feature in plants universally identifies or defines pcd. We have looked for a hallmark of pcd in animal cells, namely, DNA cleavage, in plant cells killed by infection with incompatible fungi or by abiotic means. We found that cell death triggered in intact leaves of two resistant cowpea cultivars by the cowpea rust fungus is accompanied by the cleavage of nuclear DNA into oligonucleosomal fragments (DNA laddering). Terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labeling of leaf sections showed that fungus-induced DNA cleavage occurred only in haustorium-containing cells and was detectable early in the degeneration process. Such cytologically detectable DNA cleavage was also observed in vascular tissue of infected and uninfected plants, but no DNA laddering was detected in the latter. DNA laddering was triggered by [greater than or equal to]100 mM KCN, regardless of cowpea cultivar, but not by physical cell disruption or by concentrations of H2O2, NaN3, CuSO4, or ZnCl2 that killed cowpea cells at a rate similar to that of ladder-inducing KCN concentrations. These and other results suggest that the hypersensitive response to microbial pathogens may involve a pcd with some of the characteristics of animal apoptosis and that DNA cleavage is a potential indicator of pcd in plants.

RECYCLING OF METABOLIZED IRON BY THE MARINE DINOFLAGELLATE AMPHIDINIUM CARTERAE1

ABSTRACTSenescent, iron‐limited cultures of Amphidinium carterae Hulburt were used to detect the presence of iron made available for phytoplankton growth by recycling of metabolized iron from disrupted cells. Both physical disruption of cells and physical disruption plus exposure to a low pH were tested. The test cultures remained viable over long periods of time, and were stimulated to full growth by the addition of Fe, but not by the addition of any other nutrients. Simple physical disruption of cells caused only a very slow release of available Fe, while disruption of cells plus exposure to a low pH resulted in a rapid release of available Fe. It is suggested that the digestive processes of herbivores are instrumental in the rapid regeneration of Fe as a nutrient available for phytoplankton growth.