Continuous stirred ultrafiltration

The investigation into the kinetic behavior of both enzymes was in general performed in both batch and continuous stirred ultrafiltration (UF)-membrane reac-... 

Bioreactors a) batch stirred tank b) continuous stirred tank c) continuous packed-bed i) downward flow, ii) upward flow and iii) recycle d) continuous fluidised-bed e) continuous ultrafiltration. Redrawn from Katchalski - Katzir E. (1993) Trends in Biotechnology II, 471-477. 

An excellent production figure for (R)-mandelonitrile (2400 g/1 per day) was achieved by Kragl et al. [105] using a continuously stirred tank reactor in which an ultrafiltration membrane enables continuous homogenous catalysis to occur from an enzyme (PaHnl) which is retained within the reaction vessel. In order to quench the reaction the outlet of this vessel was fed into a vessel containing a mixture of chloroform and hydrochloric acid, which allowed for accurate product analysis. 

A solution to this problem is the enzyme membrane reactor (Figure 10.8). This is a kind of CSTR (continuous stirred tank reactor), with retains the enzyme and the cofactor using an ultrafiltration membrane. This membrane has a cut-off of about 10000. Enzymes usually have a molecular mass of 25000-250000, but the molecular mass of NAD(H) is much too low for retention. Therefore it is first derivatized with polyethylene glycol (PEG 20000). The reactivity of NAD(H) is hardly affected by the derivatization with this soluble polymer. Alanine can now be produced continuously by high concentrations of both enzymes and of NAD (H) in this reactor. 

The production of substances that preserve the food from contamination or from oxidation is another important field of membrane bioreactor. For example, the production of high amounts of propionic acid, commonly used as antifungal substance, was carried out by a continuous stirred-tank reactor associated with ultrafiltration cell recycle and a nanofiltration membrane [51] or the production of gluconic acid by the use of glucose oxidase in a bioreactor using P E S membranes [52]. Lactic acid is widely used as an acidulant, flavor additive, and preservative in the food, pharmaceutical, leather, and textile industries. As an intermediate product in mammalian metabolism, L( +) lactic acid is more important in the food industry than the D(—) isomer. The performance of an improved fermentation system, that is, a membrane cell-recycle bioreactors MCRB was studied [53, 54], the maximum productivity of 31.5 g/Lh was recorded, 10 times greater than the counterpart of the batch-fed fermentation [54]. 

In order to assess the potential applications of these new nitrilases in biocatalytic processes, data on their operational stabiUty were required. To this end, we investigated the kinetic behavior of enzymes from F. solani and A. niger either immo-biUzed on solid supports or retained in stirred ultrafiltration membrane reactors in continuous experiments. 

Figure 3-3. Pressure ultrafiltration. The solution to be concentrated is placed in the ultrafiltration chamber which is fitted with a semi-permeable membrane on the lower surface, and filtered under pressure. Membrane clogging is prevented by continuous stirring of the solution.

Compared to batch processes, continuous processes often show a higher space-time yield. Reaction conditions may be kept within certain limits more easily. For easier scale-up of some enzyme-catalyzed reactions, the Enzyme Membrane Reactor (EMR) has been developed. The principle is shown in Fig. 7-26 A. The difference in size between a biocatalyst and the reactants enables continuous homogeneous catalysis to be achieved while retaining the catalyst in the vessel. For this purpose, commercially available ultrafiltration membranes are used. When continuously operated, the EMR behaves as a continuous stirred tank reactor (CSTR) with complete backmixing. For large-scale membrane reactors, hollow-fiber membranes or stacked flat membranes are used 129. To prevent concentration polarization on the membrane, the reaction mixture is circulated along the membrane surface by a low-shear recirculation pump (Fig. 7-26 B). 

Fig. 5.1 EHfferent configurations of reactors with immobilized enzymes A batch B recirculation batch C stirred tank-ultrafiltration D continuous stirred tank E continuous packed-bed F continuous fluidized-bed...

The "slurry reactor, which was introduced by Bossow and Wandrey for enzymatic formation of C-C bonds (25), is basically a stirred ultrafiltration cell (10 mL) containing a membrane with a defined molecular weight limit of 100 or 300 kD, which serves as barrier. The components of the galactosylation mixture are continuously pumped through the reactor and pass through this barrier, whereas the immobilized catalyst is retained in the reactor. The immobilized enzyme is held in suspension by continuous stirring. 

In this section, the flow vs. force configuration of a continuous stirred tank separator (CSTS) will be illustrated with a few examples. The examples cover crystallization, solvent extraction, ultrafiltration and gas permeation. 

Figure 6.4.1. Well-stirred separations (a) continuous well-stirred crystalUzer (MSMPR) (b) continuous well-stirred solvent extraction device (c) continuous well-stirred ultrafiltration cell (d) continuous well-stirred gps separation ceii.

Figure 6.4.9. Ultrafiltratton in a well-stirred vessel (a) batch ultrafiltration (b) continuous diajUtration/ultrafiltration (c) discontinuous diafiltration using three stages.

Schroer et al. developed a continuous process for the asymmetric reduction of methyl acetoacetate using iso-propanol as a cosubstrate in a continuously stirred tank reactor using an ultrafiltration membrane for retention of cells [52]. The bioreduction was run continuously for 7 weeks with exceedingly high substrate and cosubstrate concentration of up to 2.5 and 2.8 mol/L. Maximal space-time yield of about 700 g/L/day was achieved. 

The simplest ultrafiltration is the stirred cell, a batch operation. The most compex is a continuous stages-in-series operation incorporating diafiltration. Industrial practice incorporates the full gamut of complexity. 

The enzymatic system used for the continuous production of Mn3+-malonate is presented in Fig. 10.3. It is composed by a stirred tank reactor (200-mL working volume) operated in continuous mode coupled to a 10 kDa cutoff ultrafiltration membrane (Prep/Scale-TFF Millipore), which permits the recycling of the enzyme to the reaction vessel. The enzyme was recycled in a recycling feed flow ratio of 12 1. 

Thereafter, the continuous production of the complex Mn3+-malonate was carried out in a stirred reactor coupled with an external ultrafiltration membrane. The continuous reactor production of Mn3+-chelate was coupled with a degradation... 

Continuous mns were carried out in a stirred cell ultrafiltration module using the fluoropolymer membrane FS61PP with a nominal molecular weight cut-off of 20kDa. The reactor, loaded with an appropriate amount of resting cells, was fed with a buffered substrate solution by a peristaltic pump with the flow rate set at... 

The N-acetyl-D,L-amino acid precursors are conveniently accessible through either acetylation of D,L-amino acids with acetyl chloride or acetic anhydride in a Schotten-Baumann reaction or via amidocarbonylation I801. For the acylase reaction, Co2+ as metal effector is added to yield an increased operational stability of the enzyme. The unconverted acetyl-D-methionine is racemized by acetic anhydride in alkali, and the racemic acetyl-D,L-methionine is reused. The racemization can also be carried out in a molten bath or by an acetyl amino acid racemase. Product recovery of L-methionine is achieved by crystallization, because L-methionine is much less soluble than the acetyl substrate. The production is carried out in a continuously operated stirred tank reactor. A polyamide ultrafiltration membrane with a cutoff of 10 kDa retains the enzyme, thus decoupling the residence times of catalyst and reactants. L-methionine is produced with an ee > 99.5 % and a yield of 80% with a capacity of > 3001 a-1. At Degussa, several proteinogenic and non-proteinogenic amino acids are produced in the same way e.g. L-alanine, L-phenylalanine, a-amino butyric acid, L-valine, l-norvaline and L-homophenylalanine. 

In the previous examples the membranes have been considered generally as semiperineable barriers for the separation of small molecules from bigger ones. When in parallel to the separation a chemical reaction takes place in the bulk solution or in the membrane itself, the system may be identified as a true membrane reactor. A classical example is a stirred-tank enzymatic reactor connected by a continuous recirculation loop to an ultrafiltration or dialysis unit. Such a system, when well designed, permits the continuous removal of the reaction products from the bulk solution without loss of enzyme (or the insoluble or macromolecular substrate ). 

Consider a time interval dt over which a differential volume dV(, of the buffer is added to the well-stirred feed vessel containing a solution volume Vfo to start with, this solution has a solute concentration Cg, (the lower molecular weight impurity or the macrosolute of interest). Continuous diafiltration is carried out such that the solution volume in the feed vessel remains constant at V/q. Therefore the volume of ultrafiltrate produced in time dt is dVb- it has a species i concentration of Cip at time t. A molar balance on species i leads to... 

Example 6.4.10 Compare the purification achieved for a protein (species i) in relation to a low molecular weight impurity (species j) for the following two processes one-stage discontinuous diafiltration vs. continuous diafiltration. The initial volume of solution is 10 liter the amount of ultrafiltrate produced is 9.5 liter. The UF vessel is well-stirred. The values of i ,- and Rj for the membrane used are flj = 1, Rj = 0. Employ a separation factor, a,j, to compare. 

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