Rosen, Natasha Nicola; (1995) Design of a biotransformation process for dehalogenation of chlorinated compounds. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Biotransformations have traditionally been used for the synthesis of novel organic molecules but they can also be used for the degradation of toxic and environmentally harmful compounds, such as haloalkanes. Some haloaliphatic compounds used in industry are toxic, carcinogenic, and resistant to degradation and so tend to accumulate in the environment unless the halogen substituents are removed. Large-scale use of these halogenated aliphatic compounds in industry has led to widespread contamination. Rhodococcus erythropolis Y2, which utilizes many of these compounds as carbon and energy sources, possesses a dehalogenase. The biotransformation involves the conversion of the haloalkane to the corresponding alcohol by cleavage of the carbon-halogen bond; further reactions allow complete mineralization of the haloalkane. The enzyme shows great industrial potential as it is a stable biocatalyst and does not require cofactors or oxygen to function. The main objectives of this project were to demonstrate the production of the haloalkane halidohydrolase from Rhodococcus erythropolis Y2 on a large scale and to examine the use of this halidohydrolase in an immobilized form for the removal of halogenated aliphatic compounds from aqueous process streams. A small scale enzyme production process has been developed which has aimed to optimize the fermentation, enzyme induction and isolation procedures. Changes in the fermentation medium and conditions have resulted in an increase in the final cell concentration from 0.7 to 5.9 gdw/L. A less volatile more effective inducer, 4-chlorobutanol, has been found. When added to the culture at a cell concentration of 0.8 gdw/L and a substrate concentration of 0.74 mM, the specific enzyme activity increased from the previous value of 0.05 to 0.16 U/mg. The fermentation and enzyme isolation have been scaled-up to 1000 litres, resulting in an increase in the final cell concentration to 9.28 gdw/L and a three-fold increase in the specific growth rate to 0.31 h-1. By understanding the kinetics of the halidohydrolase under various conditions, the reactor design has been evaluated. The enzyme has been immobilized from clarified cell extracts onto Eupergit C beads. Under optimal conditions, this gave an activity of 0.0185 U/g dry beads and 0.5 U/g dry beads on 1-chlorobutane and 4-chlorobutanol respectively. A packed bed reactor was able to dehalogenate completely saturated solutions of 1-chlorobutane. Lower percentage conversions were obtained with 4-chlorobutanol.

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