Cell Disruption Process Research Articles

Protein extracts from microalgae and cyanobacteria biomass. Techno-functional properties and bioactivity: A review

Microalgae and cyanobacteria are photosynthetic and unicellular organisms that contain considerable amounts of proteins, lipids, carbohydrates, and polyunsaturated fatty acids, among others, with applications in the cosmetic, pharmaceutical, and food industries. These microorganisms can accumulate protein up to 70 % of total biomass depending on the microalgal strain, hence they have been regarded as an alternative protein source for the future. Microalgal proteins have important applications such as emulsifying, foaming, and gelation properties, which are important for the determination of quality and texture of foods. Some microalgal peptides possess important bioactivity with many health-benefit effects. Therefore, to maximize the production of proteins from microalgae and cyanobacteria, many protein extraction procedures have been studied to increase the economic return. They have been tested towards higher protein yields at low energy cost, the preservation of protein native properties, and lower cell debris. This later is fundamental to facilitate the subsequent purification processes so that the overall cost can be reduced. The aim of this work is to review some cell disruption processes for the extraction of protein from microalgae and cyanobacteria, considering that this step is crucial for the overall process due to the high rigidness of microalgal cell covering, which can hamper the release of proteins. It also aims at reviewing the purification techniques after cellular disruption, from conventional to more recent approaches, and finally addresses the antioxidant, antidiabetic, antihypertensive, antibacterial and other bioactive properties of microalgal protein hydrolysates and peptides.

REAP+: A single preparation for rapid isolation of nuclei, cytoplasm, and mitochondria

REAP+ is an enhanced version of the rapid, efficient, and practical (REAP) method designed for the isolation of nuclear fractions. This improved version, REAP+, enables fast and effective extraction of mitochondria, cytoplasm, and nuclei. The mechanical cell disruption process has been optimized to cerebral tissues, snap-frozen liver, and HT22 cells with remarkable fraction enrichment. REAP+ is well-suited for samples containing minimal protein quantities, such as mouse hippocampal slices. The method was validated by Western blot and marker enzyme activities, such as LDH and G6PDH for the cytoplasmic fraction and succinate dehydrogenase and cytochrome c oxidase for the mitochondrial fraction. One of the outstanding features of this method is its rapid execution, yielding fractions within 15 min, allowing for simultaneous preparation of multiple samples. In essence, REAP+ emerges as a swift, efficient, and practical technique for the concurrent isolation of nuclei, cytoplasm, and mitochondria from various cell types and tissues. The method would be suitable to study the multicompartment translocation of proteins, such as metabolic enzymes and transcription factors migrating from cytosol to the mitochondria and nuclei. Moreover, its compatibility with small samples, such as hippocampal slices, and its potential applicability to human biopsies, highlights the potential application in medical research.

Effect of hot water immersion on acute physiological responses following resistance exercise.

Purpose: Hot water immersion (HWI) is a strategy theorised to enhance exercise recovery. However, the acute physiological responses to HWI following resistance exercise are yet to be determined. Methods: The effect of HWI on intramuscular temperature (IMT), muscle function, muscle soreness and blood markers of muscle cell disruption and inflammatory processes after resistance exercise was assessed. Sixteen resistance trained males performed resistance exercise, followed by either 10min HWI at 40°C or 10min passive recovery (PAS). Results: Post-intervention, the increase in IMT at all depths was greater for HWI compared to PAS, however this difference had disappeared by 1h post at depths of 1 and 2cm, and by 2h post at a depth of 3cm. There were no differences between groups for muscle function, muscle soreness or any blood markers. Conclusion: These results suggest that HWI is a viable means of heat therapy to support a greater IMT following resistance exercise. Recovery of muscle function and muscle soreness is independent of acute changes in IMT associated with HWI.

Protein Extraction, Precipitation, and Recovery from Chlorella sorokiniana Using Mechanochemical Methods

Protein extraction, precipitation, and recovery methods were evaluated by this study using a green alga—Chlorella sorokiniana. A mechanochemical cell disruption process was applied to facilitate protein extraction from microalgal biomass. Optimization of the mechanochemical process resulted in milling conditions that achieved a protein extraction of 52.7 ± 6.45%. The consequent acid precipitation method was optimized to recover 98.7% of proteins from the microalgal slurry. The measured protein content of the protein isolate was 41.4% w/w. These results indicate that the precipitation method is successful at recovering the extracted proteins in the algal slurry; however, the removal of non-protein solids during centrifugation and pH adjustment is not complete. The energy balance analysis elucidated that the energy demand of the protein extraction and recovery operation, at 0.83 MJ/kg dry algal biomass, is much lower than previous studies using high-pressure homogenization and membrane filtration. This study concludes that mechanochemical protein extraction and recovery is an effective, low-energy processing method, which could be used by algal biorefineries to prepare algal proteins for value-added chemical production as well as to make algal carbohydrates and lipids in the residual biomass more accessible for biofuel production.

Microalgal biofuels: Pathways towards a positive energy balance

Current microalgae biofuel production pathways are still far from being energetically self-sufficient. Simulations scenarios (1 t·y−1 of biomass) under a purely energetic perspective, have revealed some bottlenecks mainly in the cultivation and metabolite recovery processes. Technologies such as intensified photobioreactors such as AlgoFilm©, infrared light filtering units, photovoltaic panels, solvent-free metabolite recovery processes, and high biomass concentration treatments at⩾145 kg·m−3 are suggested and discussed to also have a major contribution on the net energy ratio of the process. An energy-driven biorefinery approach has been proposed to increase the energy output by obtaining bioethanol and biodiesel liquid fuels, but also photovoltaic energy during the same production process. The latter may also compensate for the large amounts of energy consumed in typical operations of the wet-pathway processes like harvesting-concentration, cell disruption, or solvent recycling processes. This perspective increased the final volume of liquid biofuels up to 57%. It was found a maximum net energy ratio of 1.9 for a double biofuel process, and 8.5 if photovoltaic energy is also considered.

Highly Efficient Water-Based Extraction of Biliprotein R-Phycoerythrin From Marine the Red-Macroalga Sarcopeltis skottsbergii by Ultrasound and High-Pressure Homogenization Methods

Sarcopeltis skottsbergii is an endemic species of the southern region of South America, with R-phycoerythrin (R-PE) as an accessory photosynthetic pigment. The production of S. skottsbergii is around twenty thousand tons of dry alga per year. The evaluation of (R-PE) in a biorefinery model is still incipient in the algal biotechnology area and will be used in the food, pharmaceutical, cosmeceutical, and nutraceutical industries. This work evaluated the cell disruption and separation processes by using two green technologies, ultrasound-assisted extraction (UAE) and high-pressure homogenization (HPH), to obtain an R-phycoerythrin enriched extract from S. skottsbergii. Two-levels three-factor central composite design (CCD) and response surface methodology (RSM) were carried out to optimize the extraction conditions, including the factors for UAE (time, amplitude, and solvent) and HPH (Pressure, number of passes, and solvent). Additionally, a second-order polynomial fit was performed to fit the experimental data by the green method. HPH method was the most efficient extraction method under the conditions obtained of 100-400 MPa pressure power, 2-3 number of passes, and distilled water as solvent. Furthermore, the experimental extraction yields ranged from 4.4-5.7 mg of PE g-1 of dry biomass under the optimal extraction conditions (400 MPa; 2 passes), which agreed with the predictive yield of 4.6-5.5 mg g-1 DW. The ultrafiltration membrane used for the separation process for both methods exhibited a rejection of R-phycoerythrin concentrated at 30 KDa. Furthermore, R-phycoerythrin showed a positive correlation between the antioxidant capacity (ORAC) in the best-selected extractions. After the extraction, the same pattern was observed in Chlorophyll a and total carotenoids with DPPH. Thus, it was an attractive non-aggressive extraction alternative with biological activity of interest for formulating biotechnological products for the food industry is suggested.

Effect of Different Extraction Methods on Physicochemical Characteristics and Antioxidant Activity of C-Phycocyanin from Dry Biomass of Arthrospira platensis.

The effect of four different extraction methods on physicochemical characteristics and functionalities of chloro-phycocyanin (CP) was investigated. Swelling (S-CP), freezing and thawing (4FT-CP), ultrasonication with freezing and thawing (4FT+U-CP), and the high-pressure cell disruption (HPCD-CP) process affected CP differently, thus resulting in different levels of solubility, DPPH scavenging activity, ABTS scavenging activity, and reducing power. Among the four CPs, HPCD-CP had the highest CP content (15.3%), purity (1.66 ± 0.16), and ∆E value but the lowest ∆b value. The ζ potential of HPCD-CP (−38.8 mV) was the highest, but the average particle size of 4FT+U-CP (719.1 nm) was the highest. UV-Vis absorption spectra and fluorescence spectra illustrated that high-pressure cell disruption-assisted extraction had more profound impacts on the microenvironment of tetrapyrrole chromophores, the environment of aromatic amino acids, and the phycocyanobilin of CP. Furthermore, HPCD-CP and 4FT-CP showed higher solubility and antioxidant activities than S-CP, especially 4FT+U-CP. The results obtained in this study demonstrate that HPCD technology could obtain a food-grade C-phycocyanin product with higher CP concentration, purity, solubility, and antioxidant activity.

Recovering Microalgal Bioresources: A Review of Cell Disruption Methods and Extraction Technologies.

Microalgae have evolved into a promising sustainable source of a wide range of compounds, including protein, carbohydrates, biomass, vitamins, animal feed, and cosmetic products. The process of extraction of intracellular composites in the microalgae industry is largely determined by the microalgal species, cultivation methods, cell wall disruption techniques, and extraction strategies. Various techniques have been applied to disrupt the cell wall and recover the intracellular molecules from microalgae, including non-mechanical, mechanical, and combined methods. A comprehensive understanding of the cell disruption processes in each method is essential to improve the efficiency of current technologies and further development of new methods in this field. In this review, an overview of microalgal cell disruption techniques and an analysis of their performance and challenges are provided. A number of studies on cell disruption and microalgae extraction are examined in order to highlight the key challenges facing the field of microalgae and their future prospects. In addition, the amount of product recovery for each species of microalgae and the important parameters for each technique are discussed. Finally, pulsed electric field (PEF)-assisted treatments, which are becoming an attractive option due to their simplicity and effectiveness in extracting microalgae compounds, are discussed in detail.

Isolation, purification and cytotoxic evaluation of lutein from mixotrophically grown Chlorella sorokiniana TH01

Mixotrophic cultivation of several microalgae simultaneously utilizing two energy sources of light and organic carbon has been reported to significantly achieve higher biomass productivity than phototrophic and heterotrophic modes. In addition, microalgae have been reported as a promising alternative source of marigold for lutein production. In this work, C. sorokiniana TH01 strain was mixotrophically grown on glucose for production of biomass and lutein. Results revealed that algal biomass productivity, lutein content and productivity reached 0.86–2.08 g/L/d, 4.62–6.89 mg/g and 3.98–14.35 mg/L/d, respectively, from 10 g/L glucose, 3 g/L NaNO3 and 2% (v/v) CO2, under light intensity of 50–500 μmol/m2/s and temperature of 27 °C for 10 days of the cultivation. The purification of lutein from the algal biomass was completed by a novel process of simultaneous cell disruption and extraction and silica-based column chromatography purification, reaching lutein purity of 98%. Moreover, the algal lutein can be preserved by solvation with 0.05 wt% ascorbic acid under temperature of <4 °C in the darkness, achieving 90% stability for 120 days of the storage. Chemical structure of the purified algal lutein was fully characterized by NMR and UV–vis spectroscopy and MS spectrometry, showing similar characteristics as the standard lutein and other lutein reported in the literature. Furthermore, the analysis of antiproliferative activity of the algal lutein against human KB, HepG2, MCF-7 and SK-LU-1 cancer cell lines using SRB assay revealed that the algal lutein exhibited moderate activities with corresponding IC50 values determined of 58.07, 61.37, 71.52 and 81.69 μg/mL, respectively. Despite unexpected cytotoxicity on these targeted human cancer cell lines, the algal lutein is a high value compound for commercial applications in food, feed, pharmaceutical and cosmetic industries. The C. sorokiniana TH01 strain was demonstrated to be a promising lutein source, alternatively replacing marigold-based lutein.

Effective solvents for proteins recovery from microalgae

From an industrial perspective, the exploitation of microalgae as protein source is of great economical and commercial interest due to several attractive characteristics. However, the protein extraction efficiency is limited by the multiple layers of rigid and thick cell walls that are enriched with recalcitrant structure of cellulose. Therefore, an efficient method of cell disruption is necessary to disintegrate the cell wall and promote the release of protein contents. The conventional methods for downstream processing, e.g. disruption, isolation, extraction, concentration and purification, are energy-intensive and costly because they typically compose of several operational units. To reduce the overall process cost and establish an economical feasible process for the large-scale production of microalgae derived products, a more cost-effective and ecofriendly technique in downstream processing is in critical demand. One of the main challenges for protein extraction from microalgae cells is the recalcitrant structure of microalgae cell wall. This work aims to provide a guideline on the selection of the solvent to facilitate the proteins release during the cell disruption process. The influences of various solvent types (methanol, ethanol, 1-propanol and water) were evaluated and compared based on the protein yields. It was found that water solvent released the highest protein concentration from microalgae compared to the other tested solvents.

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.

Optimization and Comparison of Three Cell Disruption Processes on Lipid Extraction from Microalgae

This study reports on the optimization of the operating conditions using response surface methodology and a comparative study of three promising technologies of cell disruption (bead milling, microwaves and ultrasound) to increase the lipid extraction from Nannochloropsis oceanica, Nannochloropsis gaditana and Tetraselmis suecica. Central composite designs were used for the optimization of ultrasound and microwave processes. The performance of the cell disruption processes in breaking down microalgae cells is dependent on the strain of microalgae. Microwaves (91 °C for 25 min) were the most efficient for the recovery of lipids from N. oceanica, reaching a lipid content of 49.0% dry weight. For N. gaditana, ultrasound process (80% of amplitude for 30 min) was the most efficient in terms of lipid recovery (21.7% dry weight). The two aforementioned processes are ineffective in disturbing T. suecica whatever the operating conditions used. Only the bead milling process at low flow feed rate with 0.4 mm zirconia beads made it possible to extract 12.6% dry weight from T. suecica. The fatty acid profiles of N. oceanica and T. suecica are affected by the cell disruption process applied. The calculation of specific energy consumption has shown that this criterion should not be neglected. The choice of the most suitable cell disruption process can be defined according to numerous parameters such as the microalgae studied, the total lipid extracted, the fatty acids sought, or the energy consumption.

The effect of cell size on the kinetics of yeast disintegration in a bead mill

The results of modelling the effect of cell sizes on the microorganism disintegration process are presented. Experiments were conducted in a horizontal bead mill for baker's yeast Saccharomyces cerevisiae. The kinetics of the cell disruption process was investigated by optical image analysis and the release of intracellular protein by Bradford method. A large variation in the size of yeast from one production cycle has been demonstrated. A significant increase in the constant rate of the process with an increase in the range of the cell size class was demonstrated. More beneficial values of the coefficient of determination were obtained for the spherical model of the cell. Worse results of the process description were obtained for the ellipsoidal model. They may result from large variations in cell volume in individual size classes. The greatest changes in the cell Elongation parameter within one size class ranged from 1.00 to 1.54.

Lipid extraction from microalgae covered with biomineralized cell walls using liquefied dimethyl ether

Cell disruption is regarded as an indispensable pretreatment step before the extraction of microalgae with biomineralized cell walls. Here, two typical microalgae—diatom Chaetoceros gracilis (C. gracilis) and coccolithophore Pleurochrysis carterae (P. carterae)—covered by “hard” biomineralized cell walls were used as starting materials for lipid extraction using liquefied dimethyl ether (DME) without any pretreatment such as drying or cell disruption. The liquefied DME extraction experiments were performed at 25 °C and 0.59 MPa using a semi-continuous, flow-type system. The results of the yield, elemental composition, molecular weight distribution, fatty acid composition, and trace element composition indicated that the performance of liquefied DME extraction was similar to that of Bligh–Dyer extraction and better than that of hexane Soxhlet extraction, despite the latter two methods requiring pre-drying and cell disruption processes. It was also proven that the cell wall of microalgae would not affect lipid extraction of liquefied DME, thereby the liquefied DME extraction method is suitable for extracting lipids from microalgae with biomineralized cell walls. Besides, the lipids extracted by liquefied DME can be further used for biodiesel production.

Evaluation of CO2-induced azole-based switchable ionic liquid with hydrophobic/hydrophilic reversible transition as single solvent system for coupling lipid extraction and separation from wet microalgae

The utilization of microalgae as bioenergy source was limited by the excessive cost and energy consumption during the process of lipid extraction and separation. CO2-induced switchable ionic liquids (S-ILs) with reversible hydrophobic-hydrophilic conversion were synthesized and applied for lipid extraction and separation. The reversible transition mechanism of switchable IL is due to the formation of carbamate. The novel approach based on S-ILs was developed for lipid extraction from wet microalgae, which coupled microalgae cell disruption, lipid extraction, separation, and solvent recovery process without additional solvents. The highest lipid extraction efficiencies from wet microalgae were obtained by C6DIPA-Im, and the lipids were recovered from the extraction phase by simply bubbling CO2. Furthermore, C6DIPA-Im maintained more than 83.6 ± 3.6% of its initial lipid extraction efficiency after recycling five times. The S-IL based extraction and separation method provides a new strategy for sustainable bioenergy production.

Hydrodynamic cavitation for energy efficient and scalable process of microalgae cell disruption

Microalgae are likely to emerge as a sustainable source of biomass which can be utilized as food, feed, and fuel. Most of the valuable products in the microalgae are present intracellular. Microalgae cell wall is rigid and mainly composed of cellulose. Hence, it is essential to disrupt cells to extract intracellular products. Probe ultrasonication (US) and hydrodynamic cavitation (HC) were optimized for their operating parameters and various pre-treatments to obtain maximum cell disruption efficacy. Optimum conditions for maximum cell disruption using US were: solid load (1% w/v), duty cycle (80%), power input (54 W) and the US time 90 min. After pre-treatment with 0.5% w/v sodium hydroxide; the time required for the maximum cell disruption using US was reduced to 50 min. Optimum conditions for the maximum cell disruption using HC were cavitation device orifice over venturi, time (180 min), pressure (5 bar) and solid load (0.45% w/v). After pre-treatment with 0.5% w/v sodium hydroxide time required for maximum cell disruption using HC was reduced to 105 min. Further, the energy required for cell disruption using US and HC was calculated and, it was found that HC was significantly more energy efficient and scalable treatment for microalgae cell disruption.

Biodiesel synthesis from microalgae (Anabaena PCC 7120) by using barium titanium oxide (Ba2TiO4) solid base catalyst

In this study, Anabaena PCC 7120 microalgae is used as feedstock for biodiesel synthesis. Anabaena 7120 was cultivated in a closed photobioreactor. Oil was extracted by cell disruption process and purified by degumming process. Anabaena oil was characterized by GCMS spectroscopy. Barium titanium oxide (Ba2TiO4) heterogeneous catalyst was prepared by wet impregnation process and characterized through various techniques such as TGA, XRD, FTIR, HR-SEM, EDX and surface area analyzer. Basicity of synthesized catalyst was calculated by Hammett indicator titration method. The synthesized Ba2TiO4 was used in transesterification of Anabaena oil for biodiesel production and it was reused up to six cycles. The highest FAME conversion from anabaena oil was found to be 98.41% under optimized condition of 1:18 M ratio (oil:methanol), 3.5 wt% of catalyst loading and 180 min of reaction time at 65 °C temperature and 400 rpm stirring speed.

Optimization of cell culture and cell disruption processes to enhance the production of thermophilic cellulase FnCel5A in E.coli using response surface methodology

FnCel5A from Fervidobacterium nodosum is one of the most thermostable endoglucanases that have phenomenal characteristics, such as high activity, pH stability, and multi-specificity towards various substrates. However, large-scale thermophilic enzyme production is still a challenge. Herein, we focus on an optimization approach based on response surface methodology to improve the production of this enzyme. First, a Box-Behnken design was used to examine physiochemical parameters such as induction temperatures, isopropylβ-D-1-thiogalactopyranoside concentrations and induction times on the heterogeneous expression of FnCel5A gene in E. coli. The best culture was collected after adding 0.56 mM IPTG and incubating it for 29.5 h at 24°C. The highest enzymatic activity observed was 3.31 IU/mL. Second, an economical "thermolysis" cell lysis method for the liberation of the enzymes was also optimized using Box-Behnken design. The optimal levels of the variables were temperature 77°C, pH 7.71, and incubation time of 20 min, which gave about 74.3% higher activity than the well-established bead-milling cell disruption method. The maximum productivity of FnCel5A achieved (5772 IU/L) illustrated that its production increased significantly after combining both optimal models. This strategy can be scaled-up readily for overproduction of FnCel5A from recombinant E.coli to facilitate its usage in biomass energy production.

High recovery of intracellular recombinant HBsAg from Pichia pastoris via continuous pulsed laser cell disruption system optimized by response surface methodology.

In our previous study, we demonstrated that continuous power laser could be a clean, rapid, and convenient alternative to the other conventional disruption techniques for the release of recombinant hepatitis B surface antigen (rHBsAg) from Pichia pastoris. In the current work, we examined the effect of pulsed laser in the continuous laboratory-scale process on cell disruption. Design-of-experiments (DOE) methodology was used for optimization of cell disruption process to obtain the highest protein concentration in the disruption buffer. Our investigations for the pulsed laser at wavelength of 1,064nm demonstrated that for disrupting P. pastoris cell and releasing rHBsAg, the laser power was the most influential factor, and laser pulse duration and cycle number were in the second and third places. According to the results, the effect of laser power and pulse duration (time) had a direct relationship with protein concentration. For the number of cycles, however, increasing the value from the lowest point at first led to the enhancement and then reduction of protein concentration. The maximum cell disruption and rHBsAg release were recorded for the laser system in the energy input of 284mW and the pulse duration of 100mSec after four complete rounds of circulation.

Integration Process for Protein Extraction from Microalgae Using Liquid Biphasic Electric Flotation (LBEF) System

Microalgae are the most promising sources of protein, which have high potential due to their high-value protein content. Conventional methods of protein harnessing required multiple steps, and they are generally complex, time consuming, and expensive. Currently, the study of integration methods for microalgae cell disruption and protein recovery process as a single-step process is attracting considerable interest. This study aims to investigate the novel approach of integration method of electrolysis and liquid biphasic flotation for protein extraction from wet biomass of Chlorella sorokiniana CY-1 and obtaining the optimal operating conditions for the protein extraction. The optimized conditions were found at 60% (v/v) of 1-propanol as top phase, 250g/L of dipotassium hydrogen phosphate as bottom phase, crude microalgae loading of 0.1g, air flowrate of 150cc/min, flotation time of 10min, voltage of 20V and electrode's tip touching the top phase of LBEF. The protein recovery and separation efficiency after optimization were 23.4106 ± 1.2514% and 173.0870 ± 4.4752%, respectively. Comparison for LBEF with and without the aid of electric supply was also conducted, and it was found that with the aid of electrolysis, the protein recovery and separation efficiency increased compared to the LBEF without electrolysis. This novel approach minimizes the steps for overall protein recovery from microalgae, time consumption, and cost of operation, which is beneficial in bioprocessing industry.