Investigation and characterization of liquid two-phase systems for the separation of crystal mixtures by interfacial partitioning.

The interfacial partitioning behavior of ampicillin and phenylglycine crystals in different two-phase systems has been investigated. The two-phase systems employed are water/dodecane, water/1-butanol, and water/pentane/methanol. By means of partition experiments and microscopic imaging, it has been shown that the mechanism of separation strongly depends on the choice of the two-phase system. While water/dodecane features a mechanism of sheer competitive adsorption at the interface, separation in water/1-butanol is mainly due to partitioning into both liquid phases, leading to a higher degree of separation. Experiments with water/pentane/methanol have illustrated the large potential of three-component systems, as slight variations in the composition can have large effects on the separation.

Selective recovery of polyhydroxyalkanoate inclusion bodies from fermentation broth by dissolved-air flotation.

Selective dissolved-air flotation for the separation of medium-chain-length polyhydroxyalkanoate (PHA) inclusion bodies (IBs) from Pseudomonas putida cell debris is investigated. Measurements show that both P. putida cell debris and PHA IBs have an iso-electric point of approximately pH 3.5. Selective aggregation and as a result selective flotation of PHA IBs was observed near this pH. Qualitative prediction of the aggregation behaviour was possible on the basis of the Van der Waals, hydrophobic and electrostatic interactions. In some cases however, the stability of the suspension could not be explained with these forces alone. It was therefore suggested that additional interactions, such as steric/brush effects, play an important role in the aggregation process.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release.

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.

Scale-up of stirring as foam disruption (SAFD) to industrial scale.

Foam disruption by agitation-the stirring as foam disruption (SAFD) technique-was scaled up to pilot and production scale using Rushton turbines and an up-pumping hydrofoil impeller, the Scaba 3SHP1. The dominating mechanism behind SAFD-foam entrainment-was also demonstrated at production scale. The mechanistic model for SAFD defines a fictitious liquid velocity generated by the (upper) impeller near the dispersion surface, which is correlated with complete foam disruption. This model proved to be scalable, thus enabling the model to be used for the design of SAFD applications. Axial upward pumping impellers appeared to be more effective with respect to SAFD than Rushton turbines, as demonstrated by retrofitting a 12,000 l bioreactor, i.e. the triple Rushton configuration was compared with a mixed impeller configuration from Scaba with a 20% lower ungassed power draw. The retrofitted impeller configuration allowed 10% more broth without risking excessive foaming. In this way a substantial increase in the volumetric productivity of the bioreactor was achieved. Design recommendations for the application of SAFD are given in this paper. Using these recommendations for the design of a 30,000 l scale bioreactor, almost foamless Escherichia coli fermentations were realised.