K.J Ganzeveld

Effect of mass transfer limitations on the enzymatic kinetic resolution of epoxides in a two-liquid-phase system

Optically active epoxides can be obtained by kinetic resolution of racemic mixtures using enantioselective epoxide hydrolases. To increase the productivity of the conversion of sparingly aqueous soluble epoxides, we investigated the use of a two-phase aqueous/organic system. A kinetic model which takes into account interphase mass transfer, enzymatic reaction, and enzyme inactivation was developed to describe epoxide conversion in the system by the epoxide hydrolase from Agrobacterium radiobacter. A Lewis cell was used to determine model parameters and results from resolutions carried out in the Lewis cell were compared to model predictions to validate the model. It was found that n-octane is a biocompatible immiscible solvent suitable for use as the organic phase. Good agreement between the model predictions and experimental data was found when the enzyme inactivation rate was fitted. Simulations showed that mass transfer limitations have to be avoided in order to maximize the yield of enantiomerically pure epoxide. Resolution of a 39 g/L solution of racemic styrene oxide in octane was successfully carried out in an emulsion batch reactor to obtain (S)-styrene oxide in high enantiomeric excess (>95% e.e.) with a yield of 30%.

Upgrading of organic waste: production of the copolymer poly-3-hydroxybutyrate-co-valerate by Ralstonia eutrophus with organic waste as sole carbon source

Abstract Two types of fermented organic waste (trade and industry waste and fruit and vegetable waste) were successfully used as a sole carbon source to produce poly-3-hydroxybutyrate-co-valerate (PHBV) by Ralstonia eutrophus (formerly Alcaligenes eutrophus) via oxygen limitation. The production of PHBV could be optimized by optimizing the oxygen transfer through the fermentor. Thereby, a peak concentration of 1.1 g PHBV per liter cell suspension, 40 w% of cell dry weight, was obtained at an aeration rate of 0.24 mol O2/h·kg biomass. The yield of PHBV on the fatty acid concentration in the organic waste was 0.16 g polymer/g volatile organic matter. The process obtained, compares well with the commercial production process of PHBV based on glucose.

Experimental pulse technique for the study of microbial kinetics in continuous culture

A novel technique was developed for studying the growth kinetics of microorganisms in continuous culture. The method is based on following small perturbations of a chemostat culture by on-line measurement of the dynamic response in oxygen consumption rates. A mathematical model, incorporating microbial kinetics and mass transfer between gas and liquid phases, was applied to interpret the data. Facilitating the use of very small disturbances, the technique is non-disruptive as well as fast and accurate. The technique was used to study the growth kinetics of two cultures, Methylosinus trichosporium OB3b growing on methane, both in the presence and in the absence of copper, and Burkholderia (Pseudomonas) cepacia G4 growing on phenol. Using headspace flushes, gas blocks and liquid substrate pulse experiments, estimates for limiting substrate concentrations, maximum conversion rates Vmax and half saturation constants Ks could rapidly be obtained. For M. trichosporium OB3b it was found that it had a far higher affinity for methane when particulate methane monooxygenase (pMMO) was expressed than when the soluble form (sMMO) was expressed under copper limitation. While for B. cepacia G4 the oxygen consumption pattern during a phenol pulse in the chemostat indicated that phenol was transiently converted to an intermediate (4-hydroxy-2-oxovalerate), so that initially less oxygen was used per mole of phenol.

The modelling of counter-rotating twin screw extruders as reactors for single-component reactions

Numerical models are useful to study the behaviour of the extruder as a polymerization reactor. With a correct numerical model a theoretical analysis of the influence of several reaction and extruder parameters can be made, the limitations of the use of the extruder reactor can be determined and the effects of scale-up can be studied. The numerical model developed for a single-component reaction in a twin screw extruder shows good agreement with experimental data for the polymerization of n-butylmethacrylate during the reactive extrusion process. At low rotation rates a disagreement may occur between experiments and predicted data. However, the program is very useful for industrial scale processes where extruders operate in the area of high rotational speeds.

Trichloroethene degradation in a two‐step system by methylosinus trichosporium OB3b. Optimization of system performance: Use of formate and methane

The breakdown of dissolved TCE in a two-step bioremediation system is described. In the first reactor, the organism Methylosinus trichosporium OB3b is grown; in the second reactor, consisting of three 17-L column reactors in series, the cells degrade TCE. A special design allowed both for the addition of air (uG,s = 0.01-0. 04 mm s-1) in the conversion reactor to prevent oxygen limitation while minimizing stripping of TCE, and for the use of methane as exogenous electron donor. In two-step systems presented thus far, only formate was used (excess, 20 mM). We found formate additions could be reduced by 75% (15 degrees C), whereas small amounts of methane (0.02-0.04 mol CH4/g cells) could replace formate and led to equally optimal results. Example calculations show that up to 90% reduction in operating cost of chemicals can be obtained by using methane instead of formate. A model was developed to describe each of the conditions studied: excess formate and optimal methane addition, suboptimal formate addition and suboptimal methane addition. Using parameters obtained from independent batch experiments, the model gives a very good description of the overall TCE conversion in the two-step system. The system presented is flexible (oxygen/methane addition) and can easily be scaled up for field application. The model provides a tool for the design of an effective and low-cost treatment system based on methane addition in the conversion reactor.

The reactive extrusion of thermoplastic polyurethane and the effect of the depolymerization reaction

Abstract The reactive extrusion of thermoplastic polyurethane in a corotating twin-screw extruder was investigated. The polyurethane system consisted of a mixture of 2,4-diphenylmethane diisocyanate (2,4-MDI) and 4,4-MDI, methyl-propane-diol and a polyester polyol. An engineering extrusion model was designed and compared with experimental results. In this validation study the catalyst level, throughput, rotation speed and the barrel wall temperature was varied. A comparison of the experiments with the model showed that the model captured the polyurethane extrusion fairly well. Furthermore, the effect of the depolymerization reaction on the polyurethanes extrusion was investigated. It was found that the extruder operation is gravely affected by the depolymerization reaction: the depolymerization reaction limits the maximal obtainable conversion, stabilizes the extruder operation, and causes undesired post-extrusion curing of the polyurethane.

NADH-regulated metabolic model for growth of Methylosinus trichosporium OB3b. Cometabolic degradation of trichloroethene and optimization of bioreactor system performance

A metabolic model describing growth of Methylosinus trichosporium OB3b and cometabolic contaminant conversion is used to optimize trichloroethene (TCE) conversion in a bioreactor system. Different process configurations are compared: a growing culture and a nongrowing culture to which TCE is added at both constant and pulsed levels. The growth part of the model, presented in the preceding article, gives a detailed description of the NADH regeneration required for continued TCE conversion. It is based on the metabolic pathways, includes Michaelis‐Menten type enzyme kinetics, and uses NADH as an integrating and controlling factor. Here the model is extended to include TCE transformation, incorporating the kinetics of contaminant conversion, the related NADH consumption, toxic effects, and competitive inhibition between TCE and methane. The model realistically describes the experimentally observed negative effects of the TCE conversion products, both on soluble methane monooxygenase through the explicit incorporation of the activity of this enzyme and on cell viability through the distinction between dividing and nondividing cells. In growth‐based systems, the toxicity of the TCE conversion products causes rapid cell death, which leads to wash‐out of suspended cultures at low TCE loads (below μM inlet concentrations). Enzyme activity, which is less sensitive, is hardly affected by the toxicity of the TCE conversion products and ensures high conversions (>95%) up to the point of wash‐out. Pulsed addition of TCE (0.014–0.048 mM) leads to a complete loss of viability. However, the remaining enzyme activity can still almost completely convert the subsequently added large TCE pulses (0.33–0.64 mM). This emphasizes the inefficient use of enzyme activity in growth‐based systems. A comparison of growth‐based and similar non‐growth‐based systems reveals that the highest TCE conversions per amount of cells grown can be obtained in the latter. Using small amounts of methane (negligible compared to the amount needed to grow the cells), NADH limitation in the second step of this two‐step system can be eliminated. This results in complete utilization of enzyme activity and thus in a very effective treatment system.