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%.

Experimental Studies on the Carboxymethylation of Arrowroot Starch in Isopropanol-Water Media

The reaction between granular arrowroot starch and sodium monochloroacetate (SMCA) in isopropanol-water mixtures has been studied in a systematic way using experimental design strategies. The effect of six factors, i.e. the theoretical degree of substitution (DSt), reaction time, weight fraction of water in the mixture, NaOH/SMCA ratio, temperature and weight fraction of starch on three responses, i.e. the degree of substitution (DS), the conversion of SMCA and the selectivity of SMCA towards carboxymethyl starch, has been determined in a systematic manner. Granular carboxymethyl arrowroot starch with a maximum DS of 1.4 could be prepared in a single-step procedure. The results are compared with data obtained for potato starch. Similar trends for all responses were observed, suggesting close similarities between the chemical composition and the topochemistry of granular arrowroot- and potato-starch.

Exploratory Studies on the Carboxymethylation of Cassava Starch in Water-miscible Organic Media

The carboxymethylation of cassava starch using sodium monochloroacetate (SMCA) as an etherification agent was investigated. Mixtures of water and water-miscible organic liquids were selected as carboxymethylation reaction medium to obtain a high degree of substitution (DS) without changing the granular form and to prevent gelatinization. Factors that affect the process of chemical modification of cassava starch, including the type of solvents, the mass percentage of water in the reaction medium, the mass percentage of starch, the molar ratio of NaOH to SMCA, the theoretical DS (DSt), and the temperature were investigated experimentally. Isopropanol and tert-butanol appear to be the best solvents for the carboxymethylation process of cassava starch, and the optimum condition for this etherification reaction are a reaction medium consisting of about 10 wt% water, a reaction temperature between 50 and 55°C and a starch mass percentage between 4–8 wt%. When applying these conditions in combination with a DSt of 2.5, granular modified cassava starch with a DS of 1.4 can be obtained. The experimental results will be compared with those reported for other types of granular starches.

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.

NADH-Regulated Metabolic Model for Growth of Methylosinus trichosporium OB3b. Model Presentation, Parameter Estimation, and Model Validation

A biochemical model is presented that describes growth of Methylosinus trichosporium OB3b on methane. The model, which was developed to compare strategies to alleviate NADH limitation resulting from cometabolic contaminant conversion, includes (1) catabolism of methane via methanol, formaldehyde, and formate to carbon dioxide; (2) growth as formaldehyde assimilation; and (3) storage material (poly-β-hydroxybutyric acid, PHB) metabolism. To integrate the three processes, the cofactor NADH is used as central intermediate and controlling factor-instead of the commonly applied energy carrier ATP. This way a stable and well-regulated growth model is obtained that gives a realistic description of a variety of steady-state and transient-state experimental data. An analysis of the cells’ physiological properties is given to illustrate the applicability of the model. Steady-state model calculations showed that in strain OB3b flux control is located primarily at the first enzyme of the metabolic pathway. Since no adaptation in VMAX values is necessary to describe growth at different dilution rates, the organism seems to have a “rigid enzyme system”, the activity of which is not regulated in response to continued growth at low rates. During transient periods of excess carbon and energy source availability, PHB is found to accumulate, serving as a sink for transiently available excess reducing power.

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

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.