Downstream processing extracellular products

Downstream processing Intracellular products have to be extracted from the cells (by sonication, freeze thawing, and/or homogenization), and separated from the cells (centrifugation or filtration). Extracellular products require concentration and separation from the bulk supernatant. 

At harvest, the benzylpenicilhn is in solution extracellularly, together with a range of other metabolites and medium constituents. The first step in downstream processing is to remove the cells by filtration or centrifugation. This stage is carried out under conditions that avoid contamination with (3-lactamase-producing microorganisms which could lead to serious or total loss of product. 

The vast bulk of proteins synthesized naturally by E. coli (i.e. its homologous proteins) are intracellular. Few are exported to the periplasmic space or released as true extracellular proteins. Heterologous proteins expressed in E. coli thus invariably accumulate in the cell cytoplasm. Intracellular protein production complicates downstream processing (relative to extracellular production) as ... 

Targeted extracellular recombinant production generally results in low-level extracellular accumulation of the desired protein (often in the milligram per litre range). Extracellular production simplifies subsequent downstream processing, as discussed later in this chapter. 

The fermentation products can be the cells themselves (biomass), components within the fermentation broth (extracellular), or those trapped in cells (intracellular), examples of which are listed in Table 10.1. As shown in Figure 10.1, if the product of our interest is the cell, cells are separated from the fermentation broth and then washed and dried. In the case of extracellular products, after the cells are separated, products in the dilute aqueous medium need to be recovered and purified. The intracellular products can be released by rupturing the cells and then they can be recovered and purified. The downstream processing for enzyme reactions will be similar to the process for extracellular products. 

The complexity of biological processes generally requires many stages to produce a final, purified product from a particular composition of raw materials. Although a typical bioprocess consists of two main parts, upstream fermentation and downstream product recovery, it is not unusual to have between 10 and 20 steps in the overall process. This reflects the complex nature of a typical fermentation broth, which will consist of an aqueous mixture of cells, intracellular or extracellular products, unreacted substrates, and by-products of the fermentation process. From this mixture, the desired... 

In very basic terms, whether for intracellular or extracellular products, downstream processing usually involves a clarification step followed either by adsorption onto a resin, or solvent extraction followed by concentration. In... 

As can be seen from the four different examples, the choice of purification steps and procedures depends on the product location and properties and ultimately very much on the target application. Considerations are if the molecule is intracellular, cell associated, or extracellular and if it is soluble in the aqueous fermentation liquid or not. Other considerations are the fermentation medium itself and the fermentation organism. Large-scale industrial fermentation media often relies on complex, crude feedstocks as nutrient sources, as using defined nutrient sources is usually cost prohibitive. One of the major challenges is that complex fermentation media can lead to more variability in the fermentation output. To consistently deliver a robust product, downstream processing and formulation need to be able to normalize the upstream variability. 

Downstream processing includes the removal of the extracellular water (dewatering), the separation of the intracellular water (drying), the disruption of the algal cells, and the extraction of valuable components such as hpids, proteins, carbonhydrates, pigments, and hydrothermal treatments." The downstream processes account for a large part of the total production cost." ° This demonstrates the necessity to identify an optimal process for certain purpose. ... 

---