Ultrafiltration fermentation broths

Xanthans from several different sources were used in this study Xanthan samples A, B and C were kindly provided as freeze dried powder of ultrasonic degraded xanthan by Dr. B. Tinland, CERMAV, Grenoble, France. The molecular weights of these samples were determined experimentally in dilute solution by Dr. B. Tinland. Xanthan D was kindly provided as pasteurized, ultrafiltrated fermentation broth by Dr. G. Chauveteau, Institut Francais du Petrole, France. Xanthan E was kindly provided as a freeze dried sample from Dr. I. W. Sutherland, Edinburgh, Scotland. Xanthan F was obtained as a commercial, powdered material (Kelzan, Kelco Inc., a Division of Merck, San Diego CA.). Xanthan G was obtained as a commercial concentrated suspension (Flocon 4800, Pfizer, New York, NY)... 

An organic phase can be used several times provided the sample feed (fermentation broth) does contain cells or cell debris. Presence of such contaminants may render it necessary to regenerate the organic phase for its prolonged use. A literature survey suggests that the knowledge available on the recovery and reuse of surfactants is very little. However, the removal of surfactants from the stripping aqueous solution can be achieved by filtration and then can be recycled [10]. Use of ultrafiltration was also shown to be a successful technique for the separation of surfactants from reverse micellar solution [203]. 

Subtilisin BPN was prepared through a series of protein purification steps applied to the fermentation broth. These steps included ultrafiltration ethanol precipitation DEAE (diethyl-aminoethyl) Tris Acryl batch anionic exchange SP (sulfopropyl) Tris Acryl column cationic exchange and, concentration with an Amicon stirred cell. The enzyme purity was determined to be -951 via spectroscopic assays that measure the ratio of active enzyme to total protein. In addition, purity was verified via HPLC and SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 

Fermentation is typically conducted in dilute suspension culture. The low concentration in such systems limits reaction efficiency, and the presence of particulate and colloidal solids poses problems for product recovery and purification. By circulating the fermentation broth through an ultrafiltration system, it is possible to recover product continuously as they are generated while minimizing loss of enzyme or cells and keeping product concentration in the bioreactor below the self-inhibition level for the biocatalyst. This process is referred to as perfusion. As the ultrafiltration unit is part of the production process, the entire system is often considered a membrane reactor. 

In other words, how to link in a reliable way, with limited information, the macroscale process performance to local phenomena at the meso- (membrane pore) if not at the nano- (solute-solute or solute-barrier interactions) scale levels. To answer these questions, there is a need for a new methodology, based on chemical engineering principles, with a holistic approach involving well-balanced experimental/simulation/modeling parts. A first attempt to tackle this question was carried out a few years ago with a particular example the purification by ultrafiltration (UF) of a small neutral molecule from a fermentation broth, constituted of unknown peptides and proteins with sizes ranging on a very large scale [28-30]. 

We thank William DeVane for technical assistance with microfiltration and ultrafiltration operations, and John Finch and the fermentation pilot plant staff for providing fermentation broths. 

The potential of membrane separation techniques (such as cross-flow microfiltration(MF), ultrafiltration (UF), Reverse Osmosis (RO)and electrodialysis (ED) ) and membrane reactors in the treatment of fermentation broths are huge. The synergistic effects obtainable by designing the overall biotechnological process combining various membrane technique are particularly significant. 

One of the common side effects observed during extractive bioconversion is the accumulation of unwanted by-products in the system which may affect the productivity during continuous operation (14). The build up of glycerol and other non-volatile products was shown to decrease the ethanol yields during repeated fermentations in a two-phase system (12). The problem was, however, solved by dialysing the fermentation broth and also adding more yeast cells. It appears that the combination of ultrafiltration with the phase system may circumvent the problem of by-product inhibition in most of the cases. 

The filtered XlnD fermentation broth was initially concentrated into 20 mM Bis-Tris buffer at pH 6.8 in a 300 ml stirred ultrafiltration cell from Amicon (Beverly, MA) with a 30,000 Da molecular weight cut-off membrane. The protein was then bound to a Source Q anion exchange column (96 ml) and eluted over a 0 to 1 M NaCl gradient into 20 mM Bis-Tris at pH 6.8. The eluted XlnD peak was then reconcentrated and rerun on the Source Q column. From this run, the XlnD fractions were then pooled and buffer-exchanged into 20 mM acetate buffer at pH 5.0 with 100 mM NaCl on a Superdex 200 size exclusion column. The purity of the purified fiactions was assessed by SDS-PAGE, and the protein content was assessed by measuring the absorbance at 280 nm and by bicinchoninic acid (BCA) assay with bovine serum albumin (BSA) run as a standard. 

Figure 1441. Sampling from a fermenter for on-line analysis (after [366]). 1. Direct removal of fermentation broth (analyte A) 2. indirect sampling by ultrafiltration, dialysis, electrodialysis, per-vaporation, providing an analyte A of proportional concentration, normally diluted 3. indirect sampling by extraction of fermentation broth by external buffer 4. in situ measurement by means of an enzyme electrode or using a sterile housing with inserted electrode. F = fermenter, W = waste.

The use of two types of liquid membranes is described in [302] liquid emulsion membranes (LEMs), and supported liquid membranes (SLMs), where isoparaffin or kerosene and their mixtures were used as organic phases. A surfactant of the type of Span 80 served as emulsifier. LEMs are used, for example, for selective separation of L-phenylalanine from a racemic mixture of L-leucine biosynthesis as well as conversion of penicillins to 6-APA (6-aminopenicillanic acid). SLMs have a higher stability. A number of their commercial applications have been studied, e.g. in separation of penicillin from fermentation broth, as well as in the recovery of citric acid, lactic acid and some aminoacids. Compared with other separation methods (ultrafiltration, ultracentrifugation and ion exchange), LEMs and SLMs are advantageous in the separation of stereospecific isomers in racemic mixtures. 

Li Y, Shahbazi A (2006), Lactic acid recovery from cheese whey fermentation broth nsing combined ultrafiltration and nanofiltration membranes , AppZ. Biochem. Biotechnol., 129-H2,985-996. 

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