Broth clarification

Regardless of the location of the protein and its state, cell separation needs to be inemensive, simple, and reliable, as large amounts of fermentation-broth dilute in the desired product may be handled. The objectives are to obtain a well-clarified supernatant and solids of maximum dryness, avoiding contamination by using a contained operation. Centrifugation or crossflow filtration is t ically used for cell separation, and both unit operations can be run in a continuous-flow mode [Datar and Rosen, in Stephanopoulos (ed.), op. cit., pp. 369-503]. In recent years, e3q>anded-bea adsorption has become an alternative. It combines broth clarification and adsorption separation in a single step. 

Whole-broth clarification Rotary vacuum drum filtration Centrifugation Crossflow MF, UF Maintains high throughput Does not require filter aids... 

Mechanical separations of cells from fermentation broth (clarification)... 

In another study, similar fluxes have been observed. Through the use of microfiltration grade alumina membranes, broth clarification has concenuated the bacterias to a solids content of 21 % with an average permeate flux of 120 L/hr-m [Guibaud, 1989]. 

The first step following fermentation is the separation of solids from the liquid growth media, a step generally referred to as cell separation. More specifically, when the desired products are contained within the cells (intracellular) this step is called cell harvesting and when the products are extracellular it is known as broth clarification. The list of antibiotics with their producer organisms, molecular weight, and whether they are extracellular or intracellular is shown in Table 14.6. 

In many biotechnology applications, such as fermentation broth clarifications to produce common antibiotics, optimal values of TMP are in the range of 2 to 3 bar (15 to 30 psi) especially at high cell mass concentrations (> 30 wt.%) and cross-flow velocity range of 4 to 7 In the operation... 

Today many industrial fermentation broth clarifications are performed using cross-flow MF/UF membrane modules.The advantage of CFF over traditional separation processes is not only in superior product flow rates but also in higher yields or lower product losses. Using diafiltration, up to 99% recovery can be obtained.f lt ... 

Cell harvesting, broth clarification for recovery of antibiotics and hormones fi om the culture medium, for example, mycelia... 

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. 

Experiments investigated the integration of cell disruption by bead milling and product capmre by fluidised bed adsorption. By using fluidised bed adsorption, the clarification of the broth would be incorporated with the capture of the product which would result in a considerably... 

Downstream Processing Microfiltration plays a significant role in downstream processing of fermentation products in the pharmaceutical and bioprocessing industry. Examples are clarification of fermentation broths, sterile filtration, cell recycle in continuous fermentation, harvesting mammahan cells, cell washing, mycelia recovery, lysate recovery, enzyme purification, vaccines, and so forth. 

Pharmaceutical Removal of suspended matter is a frequent application for MF. Processes may be either clarification, in which the main product is a clarified liquid, or solids recovery. Separating cells or their fragments from broth is the most common application. Clarification of the broth in preparation for product recovery is the usual objective, but the primary goal may be recovery of cells. Cross-flow microfiltration competes w l with centrifugation, conventional filtration by rotary vacuum filter or filter press and decantation. MF delivers a cleaner permeate, an uncontaminated, concentrated cell product... 

The initial processing steps are determined to a large extent by the location of the product species, and they generally consist of cell/broth separation and/or cell debris removal. For products retained within the biomass during production, it is first necessary to concentrate the cell suspension before homogenization or chemical treatment to release the product. Clarification to remove the suspended solids is the process goal at this stage. 

The fermentation step to produce penicillin GA is the major cost element in the overall process to produce 6-APA. This is substantially due to the high cost of sterile engineering (Table 4.6 and 4.7). Clarification, extraction and solvent recovery steps are also significant, a reflection of the dilute and impure composition of fermentation broths. The concentration of 6-APA in the final broth has a big effect on total process costs. Thus increasing final 6-APA concentrations from 1.2-6.0% have been calculated to reduce production costs by over 50% (Table 4.8). By contrast the 6-APA production step cost is quite small, and is less that half the cost of the solvent recovery process (Table 4.6). The costs of the immobilized enzyme is not insignificant in a recent calculation it was estimated at 2.5 /kg 6-APA (Rasor and Tischer, 1998). 

Broth Samples. Clarification with Zinc Acetate-Potassium Ferrocyanide. Transfer an aliquot of filtered broth expected to contain between 5 and 15 mg. of... 

Alumina and other ceramic membranes of various microfiluaiion pore sizes have been used for the separation of yeast (saccharomyces cerevisiae) from the broth and the clarification of thin stillage [Cheryan, 1994]. A typical flux of 110 L/hr-m can be obtained with a crossflow velocity of 4 m/s and a transmembrane pressure of 1.7 bars. The crossflow velocity is found to markedly affect the membrane flux. Concenuation factors (ratios of final to initial concentrations) of 6 to 10 for both the broth and the stillage can be achieved. Backflushing with a frequency of every 5 minutes and a duration of 5 seconds helps maintain the flux, particularly in the initial operating period. The permeate flux for both types of separation reaches steady state after 30 to 90 minutes. 

Qthgr filtratjon applications. Another application is the recovery of polysaccharide from sugar fermentation broth. Good clarification results have been obtained with a flux of200] VhT-m2 [Guibaud, 1989]. 

When clarifying antibiotics there are some specific considerations to keep in mind most important is whether or not the product is intra or extracellular. Penicillin V, G, and many others are extracellular products. In such cases, the target antibiotics are excreted into the broth. For clarification, a crossflow system with ceramic membranes or hollow fibers is therefore widely used. The permeate (filtrate) contains the desired product. Diafiltration can also be used to improve upon the yield. 

If the desired product is intracellular, cells are first concentrated and then lysed to release the product. Extraction by solvent or pH change is used alone or in conjunction with the mechanical disruption. The resulting lysate is then clarified to remove cell debris leaving only the clarified products. With the introduction of ceramic membrane systems, concentration, extraction, and clarification can be done using a single system. One example of an intracellular process, i.e., vitamin B12 production (185 m broth) is shown in Figure 14.12. 

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