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The bacterial membrane is constantly remodelled in response to environmental conditions and the external supply of precursor molecules. Some bacteria are able to acquire exogenous lyso-phospholipids and convert them to the corresponding phospholipids. Here we report that some soil-dwelling bacteria have alternative options to metabolize lyso-phosphatidylglycerol (L-PG). We find that the plant-pathogen Agrobacterium tumefaciens takes up this mono-acylated phospholipid and converts it to two distinct isoforms of the non-canonical lipid bis(monoacylglycero)phosphate (BMP). Chromatographic separation and Q-TOF MS/MS analysis revealed the presence of two possible BMP stereo configurations acylated at either of the free hydroxyl groups of the glycerol head group. BMP accumulated in the inner membrane and did not visibly alter cell morphology and growth behaviour. The plant-associated bacterium Sinorhizobium meliloti was also able to convert externally provided L-PG to BMP. Other bacteria like Pseudomonas fluorescens and Escherichia coli metabolized L-PG after cell disruption, suggesting that BMP production in the natural habitat relies both on dedicated uptake systems and on head-group acylation enzymes. Overall, our study adds two previously overlooked phospholipids to the repertoire of bacterial membrane lipids and provides evidence for the remarkable condition-responsive adaptation of bacterial membranes. This article is protected by copyright. All rights reserved.

Sialic acid (SA) is an important monosaccharide that is involved in incurable cancer immunotherapy. However, it is difficult to detect SA in situ using the existing strategy based on the SA-terminated glycopeptide extraction from the cell lysate. The countermeasures of the bottleneck caused by cell disruption and peptide extraction should be designed based on a "cell-surface attachment and controlled enzymolysis" protocol. Herein, a poly(styrene-co-maleic anhydride-acrylic acid-concanavalin A) (PSM-PAA-ConA) was synthesized and developed as a pH-regulated enzyme nanoreactor after being loaded with sialidase and myoglobin. The nanoreactor showed controllable biocatalysis induced by a cascade enzyme reaction and applied for the in situ detection of SA on a living cell surface. The addition of an acidic solution resulted in a decrease in the size of the nanoreactor and enhancement of its permeability, triggering an "on" state of the SA catalysis. Subsequent pH increase led to increased hydrophilicity of the nanoreactor, increasing its size and resulting in the catalytic "off" state. ConA assisted the cell-surface attachment of the enzyme reactor. Furthermore, SA on the surface of living cancer cells was successfully monitored by the pH-regulated enzyme nanoreactor, demonstrating the feasibility of high specificity in situ analysis for SA. This pH-induced catalytic efficiency control by the enzyme nanoreactor provides a potential platform for functional stimuli-responsive catalytic systems as well as a strategy for in situ analysis of biomolecules on the cell surface.

In the present study, ozone was introduced as an alternative approach to harvest and disrupt microalgae cells (Chlorella vulgaris) simultaneously for biodiesel production. At the optimum ozonation conditions (6.14 g·h−1 ozone concentration, 30 min ozonation time, 1 L·min−1 of ozone flowrate at medium pH of 10 and temperature of 30 °C), the sedimentation efficiency of microalgae cells increased significantly from 12.56% to 68.62%. It was observed that the microalgae cells aggregated to form flocs after pretreated with ozone due to the increment of surface charge from −20 to −6.59 mV. Besides, ozone had successfully disrupted the microalgae cells and resulted in efficient lipid extraction, which was 1.9 times higher than the control sample. The extracted microalgae lipid was mainly consisted of methyl palmitate (C16:0), methyl oleate (C18:1) and methyl linolenate (C18:3), making it suitable for biodiesel production. Finally, utilization of recycled culture media after ozonation pre-treatment showed robust growth of microalgae, in which the biomass yield was maintained in the range of 0.796 to 0.879 g ·h−1 for 5 cycles of cultivation.

NAD+-dependent formate dehydrogenase from thermotolerant yeast Ogataea parapolymorpha DL-1 (OpaFDH, EC 1.2.1.2) with the additional N-terminal Gly residue and its double mutant OpaFDH_AD were overexpressed in E. coli cells. The enzyme yield was, respectively, 6000 and 6200 units per liter of the cultivation medium. Purified enzymes were obtained as homogeneous preparations with a yield of 62%. The purification procedure included ultrasonic cell disruption, heat treatment of cell free extracts at 55°C for 15 min, and hydrophobic chromatography on the Phenyl Sepharose Fast Flow. Crystallization experiments with wild-type OpaFDH resulted in the preparation of crystals of the apo-form but not the holo-form. Crystals of the holo-form were prepared in the case of the OpaFDH_AD mutant in the presence of 7 mM NAD+ and 10 mM sodium azide. The size and quality of the crystals are sufficient to collect X-ray diffraction data and determine the enzyme three-dimensional structure.

In this study, red LED and urea used as light and nitrogen sources, respectively, for the cultivation of Spirulina to enhance the fluorescence property and purity of phycocyanin. Besides, there is a high concentration of phycocyanin leached out from red light (RL) grown cells than white light (WL) without cell disruption. This type of cultivation reduces the complexity of extraction methods and cost of the downstream process. The fluorescence intensity of C-PC enhanced while using red LEDs and purity ratio improved by single-step cation exchange chromatography. Phycocyanin from red-light-exposed culture exhibited pronounced antibacterial activity against bacteria. The hydrogen peroxide scavenging activity of C-PC (93.7%) is higher than the WL cultures (88.8%). Phycocyanin from RL culture exhibited a strong antiproliferative activity (64.1%) against HeLa cancer cell line. The present study aims to analyze the influence of red light and urea on enhancing the phycocyanin production.

Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L–1 culture and a NADH oxidation activity of 0.8 µmol min−1 mg−1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii.

Metabolomics as an effective omics research tool is widely used in the study of pathogenic bacteria drug resistance mechanisms. The pretreatment methods affect the coverage and abundance of metabolites, leading to a diversity of results. Therefore, it is necessary to develop a high-coverage and robust pretreatment method to analyze and study resistant-related metabolites (carbohydrates, lipids, and amino acids). The four essential pretreatment steps of Salmonella for the GC/MS-based metabolomics study were optimized, including quenching (methanol–water and liquid nitrogen), solvent extraction (acetonitrile–isopropanol–water, methyl tert-butyl ether (MTBE) and methanol–chloroform), cell disruption (stainless-steel ball grinding and liquid nitrogen milling) and derivatization (normal-temperature and high-temperature derivatization). Metabolite profiling was performed to investigate metabolic differences between different methods. The optimized pretreatment method for the non-targeted metabolic profiling of drug-resistant Salmonella was as follows: liquid-nitrogen quenching, acetonitrile–isopropanol–water (3 : 3 : 2, v/v) as an extracting agent, stainless-steel ball grinding for cell disruption and derivatization at 37 °C. The optimized pretreatment method will provide more types of metabolites, a higher abundance of metabolites, and better repeatability and stability. The optimized method was implemented for the metabolic profiling of the resistance and sensitivity of Salmonella with serotype Derby. The method has the potential to be applied to the study of the metabolism of other pathogenic bacteria.

To facilitate the lipid extraction from Nannochloropsis oceanica with thick cell wall using switchable hydrophilicity solvent, ultrasound-assisted N, N, N', N'-tetraethyl-1,3-propanediamine (TEPDA) was used to effectively destruct the cell wall. TEPDA cations were adsorbed on the cells via electrostatic force and formed the electron-donor–acceptor (EDA) complex with the hydroxyl groups in cellulose. This broke the hydrogen-bonding interactions between cellulose chains and stripped them from cell wall, thus reducing the cell wall thickness from 141 nm to 68.6 nm. Moreover, TEPDA cations neutralized the negatively charged phospholipid bilayers, decreasing the cell surface zeta potential from −27.5 eV to −14.1 eV. The local electrostatic equilibrium led to cell membrane leakage. The ultrasound promoted the stripping of the cellulose chains at a power intensity of 0.5 W/mL and frequency of 20 kHz, achieving the lipid extraction efficiency of 98.2% within 2 h at a volume ratio of 1:4 of wet microalgae to TEPDA.

The green microalga Haematococcus pluvialis is an excellent source of astaxanthin, a powerful antioxidant widely used in cosmetics, aquaculture, health foods, and pharmaceuticals. This review explores recent developments in cell disruption and astaxanthin extraction techniques applied using H. pluvialis as a model species for large-scale algal biorefinery. Notably, this alga develops a unique cyst-like cell with a rigid three-layered cell wall during astaxanthin accumulation (∼4% of dry weight) under stress. The thick (∼2 µm), acetolysis-resistant cell wall forms the strongest barrier to astaxanthin extraction. Various physical, chemical, and biological cell disruption methods were discussed and compared based on theoretical mechanisms, biomass status (wet, dry, and live), cell-disruption efficacy, astaxanthin extractability, cost, scalability, synergistic combinations, and impact on the stress-sensitive astaxanthin content. The challenges and future prospects of the downstream processes for the sustainable and economic development of advanced H. pluvialis biorefineries are also outlined.

Natural carotenoids are secondary metabolites that exhibit antioxidant, anti-inflammatory and anti-cancer properties. These types of compounds are in high demand by pharmaceutical, cosmetic, textile and food industries, leading to the search for new natural sources of carotenoids. In recent years, the production of carotenoids from bacteria has become of great interest for industrial applications. In addition to carotenoids with C40-skeletons, some bacteria have the ability to synthesize characteristic carotenoids with C30-skeletons. In this regard, a great variety of methodologies for the extraction and identification of bacterial carotenoids has been reported and this is the first review that condenses much of this information. To understand the diversity of these carotenoids, we present their biosynthetic origin in order to focus on the methodologies employed in their extraction and characterization. Special emphasis has been made on high-performance liquid chromatography-mass spectrometry (HPLC-MS) for the analysis and identification of bacterial carotenoids. We end up this review showing their potential commercial use of bacterial carotenoids. This review is proposed as a guide for the identification of these metabolites, which are frequently reported in new bacteria strains.

The bacterial infections have been substantially increasing with higher mortality and new regimens required for their management. The present work deals with the green synthesis of silver nanoparticles (AgNPs) using leaf extract of Pteris vittata at pH 9.0. The AgNPs showed a single absorption peak at 407 nm. The morphology of AgNPs was found to be spherical in shape analyzed by scanning electron micrographs. The X-ray diffraction studies revealed the face-centered cubic structure of AgNPs with a 17-nm average crystallite size. They showed the antimicrobial activity against Pseudomonas aeruginosa, and the cell growth was completely ceased at the minimum inhibitory concentration (MIC); 100 μg/mL, with rapidly decreased cell viability. This bactericidal effect was due to the enhancement of cell permeability caused by cell disruption. The AgNPs lead to show a promising antiquorum-sensing activity by inhibition of toxin protease and pyocyanin in P. aeruginosa by 88% and, 94% respectively, at the sub-MIC concentration (0.25× MIC). These results conclude that the green synthesis of AgNPs shows a promising antimicrobial and antivirulence activity against P. aeruginosa.

In this study, the impact of different cell disruption techniques (high-pressure micro fluidization (HPMF), ionic liquids (ILs), multi-enzyme (ME), and hydrochloric acid (HCl)) on the chemical composition and biological activity of astaxanthin (AST) obtained from Haematococcus pluvialis was investigated. Results indicated that all cell disruption techniques had a significant effect on AST composition, which were confirmed by TLC and UPC2 analysis. AST recovery from HCl (HCl-AST) and ILs (ILs-AST) cell disruption techniques was dominant by free and monoesters AST, while AST recovery from HPMF (HPMF-AST) and ME (ME-AST) cell disruption techniques was composed of monoesters, diesters, and free AST. Further biological activity analysis displayed that HCl-AST showed the highest ABTS and DPPH activity, while ILs-AST showed better results against the ORAC assay. Additionally, ILs-AST exhibits a stronger anti-proliferation of HepG2 cells in a dose-dependent manner, which was ascribed to AST-induced ROS in to inhibit the proliferative of cancer cells.

BackgroundThe extraction of lipids from microalgae requires a pretreatment process to break the cell wall and subsequent extraction processes to obtain the lipids for biofuels production. The multistep operation tends to incur high costs and are energy intensive due to longer process operations. This research work applies the combination of radicals from hydrogen peroxide with an organic solvent as a chemical pretreatment method for disrupting the cell wall of microalgae and simultaneously extracting lipids from the biomass in a one-step biphasic solution.ResultSeveral parameters which can affect the biphasic system were analyzed: contact time, volume of solvent, volume ratio, type of organic solvent, biomass amount and concentration of solvents, to extract the highest amount of lipids from microalgae. The results were optimized and up to 83.5% of lipid recovery yield and 94.6% of enhancement was successfully achieved. The results obtain from GC-FID were similar to the analysis of triglyceride lipid standard.ConclusionThe profound hybrid biphasic system shows great potential to radically disrupt the cell wall of microalgae and instantaneously extract lipids in a single-step approach. The lipids extracted were tested to for its comparability to biodiesel performance.

Cell disruption is a basic yet often overlooked step in the extraction of valuable substances from plants. A simple and easily applicable method is presented to assess cell membrane stability as an integral part of phytoextraction by measuring electric conductivity (EC) and calculating the Index of Injury (Id). In this paper, a wide range of industrially applicable methods is evaluated including mechanical disruption, heat, a combination of both, extreme pH-values, application of microwaves, ultrasound and pulsed electric field (PEF). Moreover, the approach via Id is compared to an alternative method using HPLC analysis of a marker substance to show the method’s limitations as well as to provide further applications of this basic system. For the feedstock used, heavy mechanical processing such as grinding, temperatures above 50 °C as well as acidic condition below pH 2.5 were equally potent methods for complete cell disruption. Alternatively, moderate mechanical treatment like chopping or cutting could be combined with temperatures above 40 °C for maximal cell damage. Although PEF application showed merely moderate effects applied alone, it may be combined with mechanical pre-treatment in order to achieve high cell disruption at low temperatures, making it ideal for the extraction of thermolabile compounds.

Phytostilbenes are responsible for several biological activities of mulberry (Morus sp.), which has been widely used as a raw material in health products. This study aimed to investigate the capability of Morus alba L. cell in bioreactors to produce the major bioactive stilbenes. The cell obtained from air-driven bioreactors such as round bottom, flat bottom, and air-lift vessel shape bioreactors was collected and analyzed for the levels of mulberroside A and oxyresveratrol. The results showed that the cell culture in round bottom and air-lift vessel bioreactors had higher growth rate, as compared with the cell culture in shake flasks (1.38- and 1.41-fold, respectively). The optimized culture condition to produce mulberroside A was obtained from round bottom bioreactor culture (55.56 ± 11.41 μmol/L). Additionally, endogenous stilbenoid hydrolysis of cell from the bioreactor culture was examined. Under optimized hydrolytic conditions, mulberroside A in the cell was readily deglycosylated to give oxyresveratrol within 1 h. These results indicated that the glycoside mulberroside A in the cell is sensitive to the endogenous enzymatic hydrolysis. Interaction of the stilbenoid components with the endogenous hydrolytic enzyme triggered by cell disruption in M. alba samples was suggested to be the major cause of the alteration of the stilbenoid levels. These findings have provided a new approach to producing glycosidic compounds and corresponding aglycones in cell culture.

The process of extracting lipids from high-moisture Scenedesmus quadricauda microalgae biomass disrupted with microwave was examined. The study showed that microwave pre-treatment is effective in algae cell rupture while microwave power was found to be a significant factor to enhance the degree of cell disruption. Though microwave pre-treatment time had some effect, the degree of cell rupture seemed to decrease after a certain pre-treatment time. The total lipid from Scenedesmus quadricauda sp. were extracted using a mixture methanol and sulphuric acid as an organic solvent. In addition, it was discovered that microwave pre-treatment enhances the disruption of microalgae cells to attain a high level of lipid yields. Optimal lipid yield obtained in this study was 49% at power 600 W, heating time of 8 min and extraction time of 3.5 h.

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.

Spent brewer’s yeast is an excellent source of a variety of bioactive substances. In this study, for the first time, the focus was solely on investigating the influence of three industrially applicable cell disruption methods (cell mill, sonotrode, and autolysis) on selected substance groups relevant for physiology and process technology. A consistent spent yeast (Saccharomyces cerevisiae TUM 68) produced in a standardized industrial pilot top-fermenting process was used as a raw material. Using mechanical methods, i.e., cell mill and sonotrode, the protein content (as not hydrolyzed in free amino acids), the trehalose and the total fat content in the yeast extract were increased compared with those produced in the autolytic method. The analyzed B vitamin levels were also higher, the biologically active 5-CH3-H4folate in particular had the greatest proportion in the folate vitamer distribution of the mechanically produced yeast extracts. An increased level of non-fragmented genomic and mitochondrial DNA could also be found in the yeast extract produced via the mechanical methods. The antioxidative and reduction potential was decreased by the degradation of polyphenols and glutathione in the yeast extract following autolysis. The mineral, RNA, glycogen, glucose, fructose and ash contents did not differ significantly. Therefore, the cell mill and sonotrode offered a good alternative method to conventional autolytic procedures, especially to transfer physiologically relevant substance groups in higher concentrations to the yeast extract.

Microalgae-based bulk production, especially for biofuel, is not yet economically feasible. One way to improve financial performance of such systems is to maximise value through a biorefinery approach (e.g. co-production streams, waste minimisation). This can be implemented via extraction of valuable intracellular compounds upon cell disruption; however both low-cost, energy efficient, and scalable cell disruption techniques, and green chemicals are required. Waste heat is a readily available resource in most production plants, and can be used to power a hydrothermal pre-treatment (HTP) step to disrupt the cells. In this study, we use HTP (120–180 °C, 5–30 min) to disrupt Chlorella sp. cell walls. We perform a subsequent water-miscible solvent and/or urea extraction (UX) to solubilise and recover proteins. The maximum extraction efficiency of 62% was achieved after HTP at 180 °C for 10 min. Regardless of the reaction time, 120 °C was found to be ineffective for protein extraction. The efficiency can be further increased to 84% if a filter-washing step with acetone and urea is implemented. Most of the recovered proteins are between 8 to 14 kDa across all temperature ranges, suggesting that extraction efficiency depended both on partial protein hydrolysis and on accessibility to solvent (i.e. cell wall degradation, solid dispersion, membrane solubilisation). The most significant amino acid losses on a per mole basis were lysine (22%) and arginine (14%). The production of flexible and high-value protein-based products via HTP-UX may enable a circular and sustainable production step for microalgal biorefineries, while improving the downstream fuel processes through nitrogen heteroatom reduction.

Carrier-based immobilization has been developed to improve the functional properties of enzymes and promote the employment of enzymatic lysis in downstream processing without the necessity to separate the lysis enzyme after processing. In this study, lysozyme was immobilized on an extrudate-shaped Na-Y zeolite and was used for the disruption of Micrococcus lysodeikticus in a recirculating packed bed reactor. The factors influencing immobilization process, namely, liquid velocity and initial concentration of lysozyme solution, were studied aiming to improve the amount of immobilized lysozyme. Additionally, the key process parameters of cell disruption operation using lysozyme-zeolite complex in recirculating packed bed reactor, including the volume of M. lysodeikticus suspension, liquid velocity and operating temperature, were further investigated for their effects on the efficiency of cell disruption. Under the optimal conditions (50 ml of 2.0 mg/ml cell suspension, liquid velocity of 500 cm/h, and operating temperature of 25 °C), the system successfully achieved cell disruption yield of 93% and released protein content of approximately 400 μg/ml. Besides, the system demonstrated its reusability by acquiring released protein content of 250 μg/ml after 12 successive cycles of operation.

The present study elucidates the antibiofilm and antivirulent capability of glycyrrhetinic acid (GRA) against Pseudomonas aeruginosa ATCC 25619. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of GRA against P. aeruginosa were found to be 160 μg/mL and 420 μg/mL respectively. In an acclimatization resistance analysis using P. aeruginosa, no resistance towards GRA was observed during the habituation period. Adequate penetration of GRA over the biofilm matrix was proposed with the membrane penetration model assembly constructed with the preformed biofilm exhibited the prospective penetration of GRA above the mature biofilm. Furthermore, GRA resulted in the attenuation of virulence factors such as motility, biofilm formation, pyocyanin secretion, secreted proteases with its sub MIC concentrations. The antibiofilm property of GRA was assessed with the light microscopy and high content screening fluorescent imaging system, which clearly demonstrates, the thickness of P. aeruginosa biofilm was reduced to 11.33 ± 2.08 μm from 39 ± 2.51 μm. Transmission Electron Microscopy (TEM) images depicted the morphological changes in cells such as disaggregation of colonies, cell disruption with loss of intracellular material, cytolytic damage, the process of morphological transformation, bacteriolysis indicating the potential effect of GRA.

Astaxanthin (ASX) is a xanthophyll pigment considered as a nutraceutical with high antioxidant activity. Several clinical trials have shown the multiple health benefits of this molecule; therefore, it has various pharmaceutical industry applications. Commercial astaxanthin can be produced by chemical synthesis or through biosynthesis within different microorganisms. The molecule produced by the microorganisms is highly preferred due to its zero toxicity and superior therapeutic properties. However, the biotechnological production of the xanthophyll is not competitive against the chemical synthesis, since the downstream process may represent 70–80% of the process production cost. These operations denote then an opportunity to optimize the process and make this alternative more competitive. Since ASX is produced intracellularly by the microorganisms, high investment and high operational costs, like centrifugation and bead milling or high-pressure homogenization, are mainly used. In cell recovery, flocculation and flotation may represent low energy demanding techniques, whereas, after cell disruption, an efficient extraction technique is necessary to extract the highest percentage of ASX produced by the cell. Solvent extraction is the traditional method, but large-scale ASX production has adopted supercritical CO2 (SC-CO2), an efficient and environmentally friendly technology. On the other hand, assisted technologies are extensively reported since the cell disruption, and ASX extraction can be carried out in a single step. Because a high-purity product is required in pharmaceuticals and nutraceutical applications, the use of chromatography is necessary for the downstream process. Traditionally liquid-solid chromatography techniques are applied; however, the recent emergence of liquid-liquid chromatography like high-speed countercurrent chromatography (HSCCC) coupled with liquid-solid chromatography allows high productivity and purity up to 99% of ASX. Additionally, the use of SC-CO2, coupled with two-dimensional chromatography, is very promising. Finally, the purified ASX needs to be formulated to ensure its stability and bioavailability; thus, encapsulation is widely employed. In this review, we focus on the processes of cell recovery, cell disruption, drying, extraction, purification, and formulation of ASX mainly produced in Haematococcus pluvialis, Phaffia rhodozyma, and Paracoccus carotinifaciens. We discuss the current technologies that are being developed to make downstream operations more efficient and competitive in the biotechnological production process of this carotenoid.

Biodiesel produced from microalgae is a potential alternative due to the high growth rate of microalgae, the possibility of using nonarable land, and high lipid accumulation rate. Microalgae cultivation, cell harvesting and disruption are the important steps before lipid extraction for the biodiesel. In the co-submission article, the details of the whole process cannot be clearly explained. In this regard, we present the details of methods on parameter of photo-bioreactor for cultivating microalgae, flocculation tests to determine optimal flocculant dosage in harvesting, parameter of Dimethyl ether (DME) subcritical extraction device and full-factorial design for investigating the influence of extraction time, initial water content and DME dosage on the extraction performance. It will allow researchers to reproduce these experiments. • The method shows a cell disruption assisted lipid extraction by subcritical dimethyl ether. • Model is built from full-factorial design to investigate multi-factor influence. • Differential scanning calorimetry can be applicable to measure free water content., Graphical abstract Image, graphical abstract

Papain has bacteriostatic and anti-inflammatory properties and has been shown to be effective in the healing process of wounds, injuries, and ulcers by breaking down fibrinous material into necrotic tissue. However, papain is unstable and easily denatures or deactivates preventing their broad industrial application. In this work, papain was incorporated into SBA-15 ordered mesoporous silica, with the aim of improving its stability, as well as its encapsulation yield and influence of the reaction time on its stability and enzymatic activity. By small-angle X-ray scattering it was possible to verify that the papain is a globular and slightly elongated protein, likely dimeric, with a maximum size of ~6.0 nm, which favors its inclusion in the SBA-15 mesopores (~10 nm). The biocomposites (SBA-15:papain) were characterized by several physical-chemical techniques. The results indicated that the papain was incorporated into the SBA-15 mesopores. The biocomposites exhibited higher thermal degradation temperature than pure papain and did not show cytotoxic potential. The papain enzymatic activity was maintained, with the best performance for the samples prepared with 30 wt% of papain. The results also showed that the 3D cell culture system is better suited for biocomposites that present a cell disruption mechanism.

Antibiotic resistance is the phenomenon in which bacteria is or becomes resistant to antibiotics. As it pertains to oral bacteria, current methods for elimination of their presence and reduction of infection is through mechanical means, such as brushing, flossing, or swishing with an alcoholic rinse. These methods loosen the bacteria from oral surfaces or attack bacteria through cell disruption by protein denaturation and lipid membrane dissolution. One strain of oral bacteria that can cause plaque accumulation, chewing difficulty, impaired speech, and dental caries is Streptococcus mutans (S. mutans). S. mutans is a group of Gram-positive, facultatively anaerobic, cocci bacteria that can contribute to a multitude of oral hygiene concerns, including loss of dental implants in cases of high concentration. Research suggests that modification of the cell wall may lead to new developments on how to regulate cell permeation, thereby, regulation of microbial resistance mechanisms. In this work, three S. mutans stains with altered peptidoglycan layers are studied through atomic force and confocal microscopy. Surface roughness differences due to molecular glycerol phosphate alterations are quantified and compared at two positions: near the pole of the cell and within the septal region. We show AFM images of bacteria scans, including the cell wall, and quantitative surface roughness measurements at the two difference regions for each strain investigated.

Subcritical dimethyl ether, a green solvent, was used to extract lipids from microalgae. The effect of the water content on the process was firstly investigated. Secondly, microalgal samples were subjected to five cell disruptions, and the effects on raw lipid and fatty acid methyl ester, and its profile were evaluated. Among them, heating, microwave, and ultrasonic treatments greatly improved extraction. Mechanism analysis revealed the improvements by the three treatments were due to increased cell wall permeability rather than to complete cell disruption. After the extraction, microalgal cells with lipid being well-extracted were shriveled with extensive surface folds, indicating a loss of intracellular substances, but the cell structure was undamaged. As for dewatering performance, extraction process removed almost all of the free water but left bound water. Finally, the potential of the residues after lipid extraction to serve as solid fuel was evaluated by combustion characteristics and heating value calculation.

The need to describe and understand signaling pathways in live cell is seen as a primary route to identifying and developing targeted medicines. Signaling cascade is also seen as a complex communication and involves interactions between multiple interconnecting proteins. Where subcellularly and how different proteins interact need to be preserved during investigation. Furthermore, these complex events occurring simultaneously may lead to a single or multiple end point or cell function such as protein synthesis, cell cytoskeleton formation, DNA damage repair, or autophagy. There is therefore a need of real-time noninvasive methods for protein assays to enable direct visualization of the interactions in their natural environment and hence overcome the limitations of methods that rely on invasive cell disruption techniques. Forster resonance energy transfer (FRET) coupled with fluorescence lifetime imaging microscopy (FLIM) is an advanced imaging method to observe protein-protein interactions at nanometer scale inside single living cells in real-time. Here we describe the development and use of two-channel pulsed interleave excitation (PIE) for multiple protein interactions in the mTORC1 pathway. The proteins were first tagged with multiple color fluorescent protein derivatives. The FRET-FLIM combination means that the information gained from using standard steady-state FRET between interacting proteins is considerably improved by monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the presence of the acceptor is evidence for a direct physical interaction.

A novel glucose-Zn-based porous coordination polymer (PCP) was selected as a carrier of cell membranes (CMs) to fabricate CM-coated PCP (CMPCP) for rapid screening of potentially active compounds from natural products. The cell disruption and the amount of maximum CMs adsorbed on PCP were optimized according to the amount of immobilized protein. This new kind of matrix exhibited good reproducibility and stability, and was applied for fishing potentially active compounds from the extracts of Vaccinium corymbosum L. leaves (VCL). Using LC-MS/MS, chlorogenic acid and quercetin were identified as the potentially active compounds through comparison of normal and non-alcoholic fatty liver disease (NAFLD)-modeled CMPCP. Our results suggested that the proposed approach based on CMPCP was environmentally friendly, cost-effective, and convenient in terms of green porous material, stable protein loading capacity, and accessible operation process. The developed method could provide a promising platform for efficient drug discovery from natural product resources.Graphical abstract.

Glycosidases enhance flavor during wine-making by mediating the enzymatic release of aroma molecules. In order to better understand the aroma enhancement potential of Oenococcus oeni SD-2a, β-D-glucosidase (βG) activities in the culture supernatant, whole cells, and disrupted cell lysate were assessed at mid log, late log and stationary growth phase. The enzymatic activity was also compared further from cell cultures with 5 different carbon sources (glucose, cellobiose, arbutin, glucose and cellobiose, glucose and arbutin) at late log phase. Correspondingly, expression levels of 3 bgl genes, OEOE-0224, OEOE-1210, and OEOE-1569 were investigated from cell cultures of the 3 growth phases, and the 5 cell cultures with different carbon sources. Finally, the volatile aroma compounds released by O. oeni SD-2a in synthetic wines with natural glycosides were evaluated by GC-MS. Results showed βG of O. oeni SD-2a was not extracellular enzyme, and the location of it didn't change with the change of growth phase and carbon source studied. βG activities in the whole cells and disrupted cell lysate were similar and constant at the 3 growth phases. As for the carbon sources, βG activities of whole cells and disrupted lysate were positively affected by cellobiose. While arbutin displayed positive and negative effect on βG activity of whole cells and disrupted lysate, respectively. It is probably that bgl genes OEOE-0224 and OEOE-1210 were related to βG activity of SD-2a whole cells, while OEOE-1569 was responsible for βG activity of disrupted lysate. More kinds of volatile compounds and higher total concentration were released by SD-2a in synthetic wine compared with control. Thus, SD-2a showed a great potential for flavor enhancement under wine-like conditions. This study provides more information for further study of βG activity from O. oeni SD-2a.

In microalgae, photosynthesis provides energy and sugar phosphates for the biosynthesis of storage and structural carbohydrates, lipids, and nitrogenous proteins. The oleaginous alga Nannochloropsis salina does not preferentially partition photoassimilates among cellulose, chrysolaminarin, and lipids in response to nitrogenous nutrient deprivation. In the present study, we investigated whether genetic impairment of the cellulose synthase gene (CesA) expression would lead to protein accumulation without the accumulation of storage C polymers in N. salina. Three cesA mutants were generated by the CRISPR/Cas9 approach. Cell wall thickness and cellulose content were reduced in the cesA1 mutant, but not in cesA2 or cesA4 cells. CesA1 mutation resulted in a reduction of chrysolaminarin and neutral lipid contents, by 66.3% and 37.1%, respectively, but increased the soluble protein content by 1.8-fold. Further, N. salina cells with a thinned cell wall were susceptible to mechanical stress, resulting in a 1.7-fold enhancement of lipid extractability. Taken together, the previous and current studies strongly suggest the presence of a controlling mechanism that regulates photoassimilate partitioning toward C and N metabolic pathways as well as the cellulose metabolism as a potential target for cost-effective microalgal cell disruption and as a useful protein production platform.

International audience; 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.

Simple Summary Bovine respiratory diseases are a key factor reducing the productivity of cows in an industrial livestock environment. We have identified a novel lysophosphatidic acid signaling antagonist, KA-1002, which alleviates lysophosphatidic acid-mediated bovine tracheal cell disruptive tissue injury and inflammation. KA-1002 could be considered a novel therapeutic reagent candidate for maintaining respiratory health in reared cattle. Abstract The industrial livestock environment can cause stress and weakened immunity in cattle, leading to microbial infections which reduce productivity. As such, there is a need for an effective therapeutic agent that can alleviate uncontrolled destructive respiratory inflammation. We found that lysophosphatidic acid (LPA), a potent endogenous stress-induced inflammatory agent, causes respiratory tissue damage and triggers inflammation in bovine bronchial cells. LPA also inflames pulmonary bovine blood vessel cells to produce inflammatory cytokines. These findings strongly suggest that LPA is a highly important endogenous material exacerbating bovine respiratory diseases. We further identified a novel LPA-signaling antagonist, KA-1002, and showed that it alleviated LPA-mediated bovine tracheal cell disruption and inflammation. Therefore, KA-1002 could potentially serve as a novel therapeutic agent to maintain physiologically healthy and balanced conditions in bovine respiratory tracts.

Cell wall disruption is a critical challenge for algal biofuel. Here, we reported Hydrothermal treatment (HTT) of high solid content (20% w/w) Nannochloropsis sp. and its influence on extraction of lipid. Various process variables were specifically studied including reaction temperature (120–200 °C) and retention time (0–60 min). SEM and TEM images revealed the cell morphology before and after HTT. A high extraction yield (84%) of lipid was obtained at 180 °C with a retention time of 60 min, without the use of any catalysts. GC–MS revealed that the main compounds in the extracts were fatty acids, hydrocarbons and esters. UPLC-MS/MS uncovered the detailed varieties of glyceride, phospholipid and glycolipid, and demonstrated that (16:0/16:0/16:1) triglyceride (TAG) was the most abundant TAG. HTT might be a promising method to break microalgae cell wall for lipid extraction.

DNA-based monitoring of pathogens in aerosol samples requires extraction methods that provide high recovery of DNA. To identify a suitable method, we evaluated six DNA extraction methods for recovery of target-specific DNA from samples with four bacterial agents at low abundance (

Downstream processing, such as cell disruption and extraction, constitutes a key step in microalgal-based industrial bioprocesses, mainly due to high costs and environmental impact. In this context, extraction technologies need to be improved, including the use of nonconventional cell disruption techniques suitable for scale-up, such as microwave and ultrasound. Therefore, this study aimed at investigating the effects of different methods of cell disruption (microwave and ultrasound) on lipid extraction from biomass of the diatom Chaetoceros calcitrans cultured in mixotrophic conditions in a medium with natural sea water and residual glycerol, with different treatment times. Both techniques applied to the biomass were efficient; that is, the results were 24.6 ± 1.3% lipids (ultrasound for 5 min) and 24.2 ± 0.9% lipids (microwave for 40 s), with no significant differences between them (p≥0.05). Likewise, there was no significant difference regarding the chemical disruption with hydrochloric acid 2 M as control (24.2 ± 1.0%). The ultrasound method consumed less energy than the microwave method. Both cell disruption methods applied to the biomass resulted in changes in the fatty acid profiles, that is, percentages of saturated fatty acids increased from 7.7% (control) to 16.6% (microwave) and 15.5% (ultrasound), whereas polyunsaturated ones increased from 12.8% (control) to 22.8% (microwave) and 21.8% (ultrasound). Concerning monounsaturated fatty acids, percentages decreased from 79.5% (control) to 60.6% (microwave) and 62.7% (ultrasound).

Several methods including microwave, Frenchpress, autoclave, bead-beating, ultrasonication, and osmotic shock were compared to identify the most effective microalgal cell disruption method. Botryococcus braunii LB572 was cultured in 5 L flasks containing JM medium mixed with oceanic sediment extract for 13 days. Among the methods tested, enhanced lipid extraction was achieved through microwave treatment (2450MHz, 1250W at 150°C for 20 min). Oleic (C18:1), linolenic (C18:3), and palmitic acids (C16:0) were found to be the major fatty acids among the C14-C24 acids from extracted lipid. In addition, the optimal conditions of transesterification were as follows: 70 mL of methanol, 6 mL of sulfuric acid, 8 mL of chloroform, and boiling at 100°C for 30 min; 85.4% of C14-C24 FAME and 78.5% of C16-C18 FAME were esterified from transesterifiable lipids.

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch) Pharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogen-the inactive precursor of trypsin-is advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. The experiments were performed with two different pH values (5.0 and 5.9) and two constant specific growth rates (0.02 and 0.04 1/h), maintained using exponential addition of methanol. All the productivity data presented rely on an active determination of trypsin obtained by proteolysis of the trypsinogen produced. The pH of the medium did not affect cell growth, but significantly influenced specific production of trypsinogen: A 1.7-fold higher concentration of trypsinogen was achieved at pH 5.9 (64 mg/L at 0.02 1/h) compared to pH 5.0. EGFP was primarily used to facilitate detection of intracellular protein over the biosynthetic time course. Using flow cytometry with fluorescence detection, cell disruption was avoided, and protein extraction and purification prior to analysis were unnecessary. However, Western blot and SDS-PAGE showed that cleavage of EGFP-trypsinogen fusion protein occurred, probably caused by Pichia-endogenous proteases. The fluorescence analysis did therefore not accurately represent the actual trypsinogen concentration. However, we gained new experimentally-relevant insights, which can be used to avoid misinterpretation of tracking and quantifying as well as online-monitoring of proteins with the frequently used fluorescent tags.

Sludge cell disruption was generally considered as the rate-limiting step for the anaerobic digestion of waste activated sludge (WAS). Advanced oxidation processes and bio-electro-chemical systems were recently reported to enhance the hydrolysis of WAS and sludge cell disruption, while the cell-breaking processes of these systems remain unclear yet. In this study, an innovative Bio-electro-Fenton system was developed to pretreat the WAS sequentially with cathode Fenton process and anode anaerobic digestion. Significant cell disruption and dissolution intracellular organics were founded after the treatment. X-ray photoelectron spectroscopy (XPS) analysis and fourier transform infrared spectroscopy (FT-IR) spectra indicated that Gram-negative bacteria were more sensitive to free radicals yielded in cathode to induce a chain reaction that destroyed the lipid-contained outer membrane, while Gram-positive bacteria with thick peptidoglycan layer were liable to be biologically decomposed in the anode. Compared with the oxidation of organic matters in the cathode Fenton, the secretion of enzyme increased in the anode which was beneficial to break down the complex matters (peptidoglycans) into simples that were available for anode oxidation by exoelectrogens. The results also showed a possible prospect for the application of this sequential pretreatment in bio-electro-Fenton systems to disrupt sludge cells and enhance the anaerobic digestion.

This study developed a way to separate lipid, polysaccharide, protein and chlorophyll from wet Chlorella spp. (85% moisture content, wt) by using three-phase partitioning (TPP). The optimum conditions for the key steps were researched and defined as (I) cell disruption at the following conditions: biomass concentration 20 g/L, bath temperature 90 °C, duration 2.5 h, enzyme concentration 12 g/L, enzyme hydrolysis time 4 h; and (II) TPP at 40% (NH4)2SO4 (wt, %), sample solution/t-butanol 1: 2 (V/V), room temperature. The extraction yield of lipid and the poysaccharide preservation rate by the optimized procedure were 80.24%, 90.63%, respectively. The protein removal rate was 78.87%. The results provided a promising towards the industrial separation of lipid, polysaccaride, protein and chlorophyll.

Background Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored. Objective This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains. Methods Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined. Results Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density (ρ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI. Conclusion Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity.

The antioxidant capacity of cell-free extracts from Lactobacillus plantarum NJAU-01 obtained by using different cell disruption methods was determined. The efficacy of various methods, including high-pressure treatment (HIP), lysozyme combined with ultrasound treatment (LCU), glycine treatment (GLY), and frozen-thawed treatment (FAT) was studied. The results showed that the bacterial suspension in the HIP group visibly had the least intact cells per visual field compared with the other groups, followed by the LCU group. The HIP group possessed 5.12 lg CFU/mL viable bacteria, which was significantly lower than that of the other groups after cell disruption from the initial 9 lg CFU/mL. The protein concentration of the cell-free extract from the HIP group was higher than that of the other groups with abundant protein bands and band intensities. The HIP group presented significantly higher total antioxidant capacity and superoxide dismutase and glutathione peroxidase activities than the other groups. Our data suggest that HIP can be a preferable method to obtain cell-free extracts with excellent antioxidant capacity for further exploring the underlying antioxidant mechanism of Lactobacillus plantarum NJAU-01.

Ultrasound pre-treatment and protein extraction of Desmodesmus sp. and Tetradesmus obliquus biomass induced residual microalgae/polycaprolactone (PCL) biocomposites with higher viscoelastic and mechanical properties as injection mould temperature increased. This was probably associated to the promotion of microalgae proteins-PCL interactions. The PCL content required, to strengthen the biocomposites, depended on the microalgae system (20 or 10 wt% for residual Desmodesmus sp. (RD) or Tetradesmus obliquus (RT), respectively). Protein degradation was observed in RT-based systems at mould temperatures higher than 100 °C. On the contrary, a greater mould temperature induced thermal crosslinking and certain cell disruption in RD-based systems. These environmentally-friendly biocomposites are an interesting alternative for replacing petroleum plastics.

The functions and properties of fungal sheath, an extracellular polysaccharide produced by many white-rot fungi, have been studied. However, the strong adherence of the sheath to fungal hyphae had been a major impediment in preparing intracellular proteins from the fungi and analyzing their cellular responses. To overcome this issue, we developed a rapid and easy method to remove the polysaccharide sheath using a selective lignin degrader, Ceriporiopsis subvermispora, which produces large sheath amounts in the presence of a lignin-derived aromatic compound. Using this approach, we achieved thorough removal of sheath and cell disruption using beads and a solution with a high protein-solubilizing power, which enabled the efficient extraction of intracellular proteins from C. subvermispora surrounded by sheath. In addition, for proteomic analysis, we investigated whether these extracted proteins were compatible with two-dimensional electrophoresis. By efficiently concentrating on protein solubilization in the first dimension and using a stacking gel in the second dimension, we successfully obtained a high-resolution proteome map of C. subvermispora. We also used the same proteins for fluorescence two-dimensional difference gel electrophoresis to obtain the quantitative protein expression profiles. These steps demonstrated that two-dimensional electrophoresis-based proteomics can be used to clarify the composition of intracellular proteins from sheath-producing white-rot fungi.

In this study, ozone was used as a pretreatment method to improve the yield in enzymatic saccharification of microalgal carbohydrates by increasing the disruption of microalgal cells. Different ozone doses (0.25-2.0 g O-3 g(-1) dry weight biomass (DWB)) were applied to the algal suspensions before the enzymatic hydrolysis process, which was performed at various enzyme concentrations, enzymatic hydrolysis times and biomass concentrations. The highest glucose yield obtained was 80.6% (w/w) of total carbohydrates under the conditions of 2.5 g L-1 biomass concentration and 4 h hydrolysis time for 0.5 g O-3 g(-1) DWB ozone dose and 1.2 mL(-1) g(-1) DWB enzyme concentration. In addition to ozone pretreatment, the same algal culture was subjected to acid, alkaline and ultrasonic pretreatments. It was observed that application of ozone increased the glucose yield much higher than the other three pretreatment methods, indicating its great potential in production of bioethanol from microalgae. (C) 2017 Elsevier Ltd. All rights reserved.

Novel third-generation polyanionic carbosilane dendrons with sulfonate or carboxylate end-groups and functionalized with a DO3A ligand at the focal point, and their corresponding copper complexes, have been prepared as antiviral compounds to prevent HIV-1 infection. The topology enables the compound to have an excellent chelating agent, DO3A, while keeping anionic peripheral groups for a therapeutic action. In this study, the cytotoxicity and anti-HIV-1 abilities of carboxylate- (5) or sulfonate-terminated (6) dendrons containing DO3A and their copper complexes (7 or 8) were evaluated. All compounds showed low cytotoxicity and demonstrated potent and broad-spectrum anti-HIV-1 activity in vitro. We also assessed the mode of antiviral action on the inhibition of HIV-1 through a panel of different in vitro antiviral assays. Our results show that copper-free dendron 6 protects the epithelial monolayer from short-term cell disruption. Copper-free dendrons 5 and 6 exert anti-HIV-1 activity at an early stage of the HIV-1 lifecycle by binding to the envelope glycoproteins of HIV-1 and by interacting with the CD4 cell receptor and blocking the binding of gp120 to CD4, and consequently HIV-1 entry. These findings show that copper-free dendrons 5 and 6 have a high potency against HIV-1 infection, confirming their non-specific ability and suggesting that these compounds deserve further study as potential candidate microbicides to prevent HIV-1 transmission.

Nowadays microbial lipids are emerged as a promising biodiesel feedstock. The recovery of intracellular lipids from oleaginous yeasts is an important step for viable application of this new lipid source. This work evaluated six different techniques for cell wall disruption and supercritical fluid assisted by ultrasound (SFE + US) for lipids extraction from oleaginous yeast cultivated in crude glycerol. Among the disruption techniques (which efficiencies ranged from 12 to 125% of the standard method), shear abrasion showed higher efficiency, as qualitatively confirmed by scanning electron microscopy images. SFE + US using CO 2 as solvent was performed for lipid extraction in the whole or previous disrupted cells, at different extraction process conditions. The global yield of SFE + US lipid extraction was statistically equal for three pressures studied with maximum of 4.96% (w/w). However, when a disruption technique was previously applied (shear abrasion), the global yield was 6.17% (w/w). The lipid profile was properly for biodiesel application and showed variable results by using different cell disruption techniques. The different SFE + US conditions did not show influence on fatty acid composition. Then this microbial lipids recovery process presented a promising environmentally friendly alternative.

Microalgae are natural renewable sources providing attractive high-value products such as proteins. Due to the high costs incurred during drying processes, wet extraction from fresh microalgae biomass can be a promising approach. In this work, a single process step integrating cell disruption and protein recovery from wet Chlorella vulgaris by using ultrasonication and aqueous solutions of ionic liquids (ILs) as extractive solvents was proposed. The ILs assessed belong to the group of Good’s buffer ionic liquids (GBILs), and were prepared by a combination of Good’s buffer-based anions, namely 2-hydroxy-3-morpholinopropanesulfonate and 2-[bis(2-hydroxyethyl)amino]ethanesulfonate, and tetrabutylammonium, tetrabutylphosphonium and cholinium cations. These GBILs possess high biocompatibility and buffering capacity at neutral pH region. The protein recovery capabilities of the aqueous solutions of GBILs were compared with conventional buffers, using the same cell disruption approach. After the proper selection of the buffer solution with higher capability to extract microalgal proteins, alternative disruption techniques such as freeze-thaw, non-ionic detergent-based lysis using Triton X-100 and the combined methods were studied. The most efficient strategy, namely ultrasonication-assisted extraction, was further investigated for the effects of the process variables such as the buffer concentration, the addition of NaCl, the biomass concentration, and the ultrasonic time and power.

Pulsed electric field (PEF) is considered to be a very promising technology for mild cell disruption. The application of PEF for microalgae that have a rigid cell wall, however, is hampered by the presence of that rigid outer cell wall. A cell wall free mutant of C. reinhardtii was used to mimic pretreated microalgae with removed cell wall, to investigate the possibility of using PEF for protein release from microalgae. A complete release of hydrophilic proteins from the cell wall free mutants was observed whereas PEF treatment on the cell wall containing species resulted in substantially lower protein yields. Additional experiments showed that even at low energy input (0.05 kWh/kgbiomass), still about 70% of the proteins could be released with respect to bead beating as reference. These released proteins were water-soluble while the hydrophobic chlorophyll remained mainly entrapped in cell particles. SEM-analysis of these cell particles showed that PEF only opened the cells, instead of completely fragmenting them into smaller particles. These results indicate that PEF is an energy-efficient cell disruption method for selective release of water-soluble proteins, after the microalgal outer cell wall is removed. Enzymatic pretreatment to degrade the cell walls before PEF treatment was shown to be an efficient method to remove the cell wall., Enzyme-assisted pulsed electric field treatment is developed as novel approach for mild, selective protein release in a multiproduct microalgae biorefinery.

High-energy and cost-intensive cell disruption processes represent one of the major techno-economic bottlenecks in the microalgae-based bio-refineries. Therefore, a feasible disruption method is required to ensure low energy input and operating cost, as well as high target-product (e.g., lipid) recovery. In this study, several different pre-treatment strategies for the disruption of Chlorella vulgaris were investigated, including NaOH/urea, sulfuric acid and ultra-sonication. Experimental results showed that the pre-treatment by NaOH/urea solution resulted in an average mass loss of 33.7 wt.%, and resulted in the removal of 77.2% of carbohydrates and 46.3% of protein (as N) from the original biomass. While these results were comparable to those obtained from the other cell disruption methods, the NaOH/urea method is believed to be more advantageous in terms of energy-efficiency and cost. Afterwards, all pre-treated microalgae samples were subjected to the liquefaction process towards bio-crude oil production. The bio-crude oils obtained from NaOH/urea solvent pre-treated microalgae resulted in higher yields and demonstrated better flow properties.