Fermentation broths, protein recovery

Initially fermentation broth has to be characterised on the viscosity of the fluid. If the presence of the biomass or cells causes trouble, they have to be removed. Tire product is stored inside the cells, the cells must be ruptured and the product must be freed. Intracellular protein can easily be precipitated, settled or filtered. In fact the product in diluted broth may not be economical enough for efficient recovery. Enrichment of the product from the bioreactor effluents for increasing product concentration may reduce the cost of product recovery. There are several economical methods for pure product recovery, such as crystallisation of the product from the concentrated broth or liquid phase. Even small amounts of cellular proteins can be lyophilised or dried from crude solution of biological products such as hormone or enzymes.2,3... 

Fluidised beds have been used previously for the industrial-scale recovery of the antibiotics streptomycin and novobiocin.30 However, more recently, considerable interest has been shown in the use of fluidised beds for the direct extraction of proteins from whole fermentation broths.31 In a packed bed, the adsorbent particles are packed within the contactor. The voidage, that is, the inter-particle space, is minimal and thus feedstock clarification is mandatory to avoid clogging of the bed. In a fluidised/expanded bed, the adsorbent bed is allowed to expand by irrigation with feedstock. Bed voidage is increased, allowing the passage of particulates in the feed. The diameters of the adsorbent beads are exaggerated for illustrative clarity. 

Morton PH, Lyddiatt A (1992) Direct recovery of protein products from whole fermentation broths A role for ion exchange adsorption in fluidized beds. In Slater MJ (ed) Ion exchange advances. Elsevier, London... 

An example of an expanded bed process for recovery of a Pseudomonas exotoxin from an E. coli system has been recently reported in pilot plant scale.28 A single-step recovery of a secreted recombinant protein has been carried out in expanded-bed mode directly from the fermentation broth without prior cell removal. The fusion protein was designed to have relatively low isoelectric point to enable anionic exchange adsorption at pH 5.5 where most of the E. coli host proteins are not adsorbed. The gene product was secreted to the culture medium of E. coli in high yield and the recovery of the protein was 90% in one step.29... 

The product may be located inside a microorganism (intracellular) or outside in the growth medium (extracellular), or alternatively, the product could be the whole cell material. The nature of the product may be solid or dissolved in the aqueous phase. For example, the product is found in the aqueous phase for a fuel ethanol fermentation, within the cell for a therapeutic protein, while the product is the whole cell in the case of single cell protein. The location of the product influences the choice of a bioseparation method which may favor the efficient recovery of either the solid or liquid phase. The relative difficulty of separating intracellular products from other unwanted insoluble materials may influence the subsequent processing steps once the solids phase has been recovered from the fermentation broth. 

Carsten Jacobsen (Novo Nordisk) presented results on protein crystallization in preclarified, concentrated fermentation broths. In particular, the impact of filtration rate on the formation of favorable large diamond versus rod shapes was examined. By adding seed crystals just above the solubility curve, where no nucleation occurred, the authors were able to produce 30% larger crystals as compared to an unseeded crystallization. Although there was minimal recovery and characterization data, this technique may prove very beneficial for dealing with difficult feed streams. While the work presented in this talk was done at the laboratory scale, scale-up experiments will be required to confirm the suitability of this approach for industrial process applications. 

The ability of proteins to transfer from an aqueous solution to a reversed micelle=icontaining organic phase, and be subsequently recovered in a second aqueous phase, was first established by the group of Luisi (2,3). It has since been suggested by van t Riet and Dekker (4, 5) and Goklen and Hatton (6 -9 ) that this phenomenon be exploited in the recovery, separation and concentration of bioproducts from complex aqueous mixtures. In the past three years, significant progress has been made in this direction, and it has been established that these solvents can be selective in the separation of binary and ternary protein mixtures (7f9 ) and in the recovery of an extracellular alkaline protease from a clarified fermentation broth (1 0). It has also been demonstrated that the process can be operated on a continuous basis (5). 

Reverse micelles are self-organized aggregates of amphiphilic molecules that provide a hydrophilic nano-scale droplet in apolar solvents. This polar core accommodates some hydrophilic biomolecules stabilized by a surfactant shell layer. Furthermore, reverse micellar solutions can extract proteins from aqueous bulk solutions through a water-oil interface. Such a liquid-liquid extraction technique is easy to scale up without a loss in resolution capability, complex equipment design, economic limitations and the impossibility of a continuous mode of operation. Therefore, reverse micellar protein extraction has great potential in facilitating large-scale protein recovery processes from fermentation broths for effective protein production. 

Recovery of Largomycin F-II. Largomycin F-II is a chromoprotein of approximately 30,000 MW produced by Streptomyces pluricolorescens. In addition to largomycin F-II, several other biologically active proteins, ranging in apparent molecular weight from less than 1,000 to greater than 400,000 are present in the fermentation broth. 

Electrodialysis uses stacks of pairs of anion- and cation-exchange membranes in deionizing water and in recovery of formic, acetic, lactic, gluconic, citric, succinic, and glutamic acids from their sodium and potassium salts in fermentation broths.114 This may have an advantage over processes that involve purification through calcium salts. Electrodialytic bipolar membranes have been used to recover concentrated mineral acids from dilute solution.115 They can be used to convert sodium chloride to hydrogen chloride and sodium hydroxide in a process that avoids the use of chlorine.116 Soy protein has been precipitated by... 

Protein Purity. During the production of recombinant proteins, process monitoring is required to assure purity levels required in every step. The recovery and purification of product from fermentation broth typically involve various procedures, such as filtration, centrifugation, and chromatography. After each purification and final step, constituent levels must be determined to ensure that the desired levels of purity have been achieved. In addition to its control function, this purity information is also frequently used to further optimize purification processes. CZE and SDS-CGE can be mostly used for the purity checks of protein products. 

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