The proposed diploma thesis aims to study the biotechnological production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer. The subject of the experimental part was first to select a suitable bacterial strain from five selected microorganisms with different carbon precursors applied at various concentrations. The five selected microorganisms used in the experimental part include bacterial strains Cupriavidus malaysiensis DSM 19416, DSM 19379, and DSM 25816. Furthermore, the strain Thermomonas hydrothermalis DSM 14834 and Aneurinibacillus thermoaerophilus H1 CCM 8960. The experiment shows that the most suitable candidate for biotechnological production is the bacterial microorganism Cupriavidus malaysiensis DSM 19379. Finally, the biotechnological production of the copolymer was investigated utilizing a batch cultivation technique in a laboratory bioreactor.

In this project a LC-MS-method for the analysis of gamma-hydroxybutyrate, gamma-butyrolactone, 1,4-butanediol, amphetamine and methadone was developed. Initially, the efficiency of the ionisation of the analytes was evaluated with respect to the ionisation technique (ESI, APCI and APPI) and the composition of the mobile phase. In the next step a number of different columns was tested in order to find the one with the greatest potential for separation of the substances in question. Using the selected column, the separation was optimised by means of experimental design and the software The Unscrambler 7.8. The parameters studied were the flow rate, the column temperature and the mobile phase composition. The response variables were the resolution between the target compounds and the retention time of the last eluting compound. These experiments showed that, in order to obtain the best ionisation, the mobile phase should consist of 5 mM formic acid in water and acetonitrile. ESI should be used in the positive mode for all analytes except gamma-hydroxybutyrate, for which the negative mode should be applied. The Hypercarb column exhibited superior retention of the analytes and was therefore selected for further optimisation. The dimensions of this column were 2.1 x 50 mm and the particle size 5 μm, connected to a 2.1 x 10 mm precolumn containing the same packing material. The optimum of the flow rate and the column temperature were 250 μl/min and 20 ºC respectively. For the separation of gamma-hydroxybutyrate, gamma-butyrolactone and 1,4-butanediol the mobile phase consisted of 100% water with 5 mM formic acid. Thereafter a gradient, up to 70% acetonitrile with 5 mM formic acid, was used in order to elute amphetamine and methadone. Efforts were also made to find an internal standard for the method. However, none of the compounds tested was found suitable. In order to get the method usable for routine analysis, which is the goal, further work is required. A suitable internal standard needs to be added to the method and thereafter work remains with validation of the method.

<p>In this project a LC-MS-method for the analysis of gamma-hydroxybutyrate, gamma-butyrolactone, 1,4-butanediol, amphetamine and methadone was developed.</p><p>Initially, the efficiency of the ionisation of the analytes was evaluated with respect to the ionisation technique (ESI, APCI and APPI) and the composition of the mobile phase. In the next step a number of different columns was tested in order to find the one with the greatest potential for separation of the substances in question. Using the selected column, the separation was optimised by means of experimental design and the software The Unscrambler 7.8. The parameters studied were the flow rate, the column temperature and the mobile phase composition. The response variables were the resolution between the target compounds and the retention time of the last eluting compound.</p><p>These experiments showed that, in order to obtain the best ionisation, the mobile phase should consist of 5 mM formic acid in water and acetonitrile. ESI should be used in the positive mode for all analytes except gamma-hydroxybutyrate, for which the negative mode should be applied. The Hypercarb column exhibited superior retention of the analytes and was therefore selected for further optimisation. The dimensions of this column were 2.1 x 50 mm and the particle size 5 μm, connected to a 2.1 x 10 mm precolumn containing the same packing material. The optimum of the flow rate and the column temperature were 250 μl/min and 20 ºC respectively. For the separation of gamma-hydroxybutyrate, gamma-butyrolactone and 1,4-butanediol the mobile phase consisted of 100% water with 5 mM formic acid. Thereafter a gradient, up to 70% acetonitrile with 5 mM formic acid, was used in order to elute amphetamine and methadone. Efforts were also made to find an internal standard for the method. However, none of the compounds tested was found suitable.</p><p>In order to get the method usable for routine analysis, which is the goal, further work is required. A suitable internal standard needs to be added to the method and thereafter work remains with validation of the method.</p>