Solvent cell disruption

Microbial fermentation broths will contain cells and cell debris, and the compound of interest may be located in solution in the broth supernatant or associated with the cell (either boimd to the cell surface or contained intracellularly). If the metabolite is cell surface-associated, measures must be taken to release/sol-vate the target compound, after which cells and cell debris may be removed by filtration or centrifugation. Addition of a water-miscible organic solvent such as methanol or acetonitrile to whole broth is a good technique for liberating cell-bound metabolites into solution. Alternatively, whole fermentation broth may be lyophilized, then the solid residue extracted with a suitable organic solvent. Cell disruption might be required for the release of intracellular metabolites. 

In general, the physical structure of the tissue must be broken down mechanically followed by an extraction procedure, before the sample can be analyzed. Homogenization using blenders, probe homogenizers, cell disrupters, sonicators, or pestle grinders is particularly useful for muscle, liver, and kidney samples. Regardless of the method used for tissue disruption, the pulse, volume of extraction solvent added, and temperature should be validated and standardized in order to ensure reproducible analytical results. During cell disruption, care should be taken to avoid heat build-up in the sample, because the analyte may be heat labile. 

Chemical Methods Chemical methods of cell rupture include the treatment of cells with detergents (surfactants), alkalis, organic solvents, or by osmotic shock. The use of chemical methods requires that the product be insensitive to the harsh environment created by the chemicals. After cell disruption, the chemicals must be easily separable or they must be compatible with the products. 

Intracellular Products. Intracellular production of bioproducts is less preferable but sometimes the only way to produce certain compounds in appreciable amounts. In this case, cell disruption is required for recovery. High-pressure homogenization, bead mills, and chemical or enzymatic disruption of the cell wall with lysozyme or similar enzymes can be used to achieve cell breakage. In the case of small molecules, organic solvent extraction has also been described. If cell debris remains in the centrate, it must be removed by methods described above, thus adding extra steps to the process. 

For the preparation of carbonyl-free solvents see Section 5.3.2.2.) The cells are washed three times in serum-free medium and harvested by scraping. The harvested cells are washed again with PBS, pH 7.4. Cell disruption is effected by light sonication and after centrifugation (600 x g, 15 min) the insoluble material is removed. The supernatant fraction is retained and the protein concentration assessed by the Lowry assay. 

Whole cells Inexpensive Enzyme co>factors present Large glassware required Messy work-up Side reactions can interfere or dominate substrate and/or product, and/or co-solvent may disrupt (membrane-bound) enzymes... 

PHA is accumulated intracellularly in Gram-negative bacterial strains and normally its recovery after the fermentation include several steps - briefly these are (i) the separation of cells from the fermentation broth by centrifugation (ii) after that, the bacterial cells are pre-treated by heat, freeze dried, or salted, before extraction to avoid polymer degradation (iii) the PHA is therefore extracted, normally by using chlorinated solvents or other methods such as enzymatic digestion or mechanical cell disruption and (iv) PHA purification. The process of PHA recoveiy is shown in Figure 2.9. 

Cell disruption methods can be roughly divided into two categories mechanical ones, e.g., bead mill, homogenizers, and ultrasound and nonmechanicals like organic solvents, osmotic shock, supersonic fluid feed (SFF) and pulsed electric field (PEF). For extracting microalgal components, several methods were applied (Table 2.6). 

Despite the several conventional methodologies used, new techniques have been developed and used in the extraction of polysaccharides and oligosaccharides aiming at an increase of the extraction yields, modification of their structure, as well as removal of other compounds that are usually destroyed during the traditional extraction process. Traditional extraction methods include hot or cold water treatments in which the variation of temperature and time of extraction are optimized to increase the extraction yields. Recently, microwave and ultrasonic extraction have been used for recovering the polysaccharides from different plant materials, mainly due to their capacity to increase cell disruption, mass transfer, and solvent penetration.Table 19.2 shows some examples of the methodologies used for the extraction of polysaccharides from different sources of plants and seaweeds. 

Cell disruption (solvents, hydrocyclone, ball mill etc.) ... 

Physical and mechanical methods are more suitable for cell disruption in general, because these methods cost less and do not affect intracellular biopolymer chemical integrity. Depending on the chemical method, temperature and material, it may affect chemical integrity if no judicious choice of solvents and operating conditions is made. Physical methods can be ultrasound, hydrocyclone, mill balls, Hughes press or osmotic pressure. 

First of all, the most efficient acid and organic solvent in disrupting the yeast cell wall and extracting astaxanthin were selected. Four acids were checked hydrochloric. 

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