Fermentation broth clarification

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

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 ... 

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. 

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. 

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. 

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). 

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]. 

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. 

Clarification With or without cell lysis, the fermentation broth consists of a mixture of components from which the final product needs to be clarified. Primary steps in clarification usually involve centrifugation and filtration to remove cell debris and other particulates. The product purity in the outlet stream is usually still low, between 1% and 10% (w/v), at the end of these steps. 

With the exception of the precoat applications, RVF s do not usually yield absolutely clear filtrate. Although still widely used, rotary vacuum filters are, in some cases, being replaced by membrane separation technology as the method of choice for clarification of fermentation broths and concentrating cell mass. Membranes can yield more complete filtration clarification, but often a wetter cell paste. 

Temperature and Viscosity. The operating temperature can have a beneficial effect on flux primarily as a result of a decrease in viscosity.f There is an additional benefit for shear thinning viscoelastic fluids, where the viscosity reduces with an increase in shear (i.e., cross-flow velocity). Typical examples are clarification of fermentation broths and concentration of protein solutions. l l It must be noted that for most fermentation and biotechnology related applications, temperature control is necessary for microbial survival and/or for product stability (e.g., antibiotics, enzymes, proteins and other colloidal materials). 

Fermentation broths tend to be very dilute and contain complex mixtures of inorganic or organic substances. The recovery of a soluble product (MW range 500-2500 dalton) such as an antibiotic, organic acid or animal vaccine from fermentation broth takes several processing steps. The first step is the clarification of broth to separate the low molecular weight soluble product from microorganisms and other particulate matter such as cells, cell debris, husks, colloids and macromolecules from the broth me-dium.l l In this step, microporous membrane filters (MWCO 10,000 to... 

Table 6.5. Comparative costs (US ) analysis for clarification methods of a fermentation broth. Adapted from Leach et al [6.19].

Initial liquid cultures in, e.g., 500-mL shake flasks can provide a sufficient volume of fermentation broth for the generation of growth and production profiles and for preliminary isolation experiments relating to clarification, stability, solvent solubility, and affinity for chromatographic resins. 

MUcent S, Carrere H. Clarification of lactic acid fermentation broths. Sep Puri Technol 2001 22 393-401. Liu C, Liu Y, Liao W, Wen Z, Chen S. Simultaneous production of nisin and lactic acid from cheese whey optimization of fermentation conditions through statistically based experimental designs. Appl Biochem Biotechnol 2004 114 627-38. 

Westoby, M., J. Chrostowski, P. de Vilmorin, J. P. Smelko, and J. K. Romero. 2011. Effects of Solution Environment on Mammalian Cell Fermentation Broth Properties Enhanced Impurity Removal and Clarification Performance. Biotechnology and Bioengineering 108 (l) 50-58. 

Milcent S, Cariere H. Clarification of lactic acid fermentation broths. Sep PuriTechnol 2001 22 393-401. 

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