On-line Monitoring Of Methanol And Glycerol In Fermentation Broth Of Pichia Pastoris By Chemiluminescence Using Sequential Injection Analysis With An Immobilized Enzyme Column

In this dissertation, a fully automated procedure based on chemiluminescence using sequential injection analysis with an immobilized enzyme column was developed and used for on-line monitoring of methanol and glycerol in fermentation broth of Pichia Pastoris. By combining the SIA time-based sampling with subsequent zone sampling approach, which allowed to select for the detection of a well-defined sample zone, it was possible to implement an on-line dilution strategy the enabled the expansion of analytical working range of the methodology, and thus its application in monitoring the key substrate in the fermentation process.The dissertation is divided into two sections.In the first section, a sequential injection analysis system is described that incorporates an enzyme column containing immobilized alcohol oxidase, allowing on-line monitoring of methanol in fermentation broth of pichia pastoris. Luminol chemiluminescence was used for the detection of the hydrogen peroxide generated in the enzyme reaction. Sample filtration and dilution were carried out on-line. The calibration graph was linear in the range of methanol concentration 0-1.0%(V/V)(R=0.9995). The relative standard deviation (R.S.D.) for the determination of methanol at a concentration of 0.2%(V/V) was 1.9% (n=11) and 5.0% during a 72-h monitoring process. The system was characterized by short analysis time, simple operation and good stability, which could meet with the requirements of on-line monitoring of methanol in fermentation process.In the second section, glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) were immobilized together on the controlled pore glass, which was packed into a mini column. After on-line pretreatment, including filtration and dilution, samples containing glycerol passed through the column. Hydrogen peroxide generated after the on-line biochemical reaction was detected by luminol chemiluminescence using sequential injection analysis. The calibration curve was linear over the range 0%-5% (V/V)(R=0.9997). The R.S.D. for the determination of glycerol at a concentration of 2%(V/V) was 2.5%(n=11). The stability of described systems was sufficient for monitoring and control of fermentation process. The enzyme column was stable for several months. This method could be applied for the monitoring of glycerol in fermentation broth of Pichia Pastoris with satisfactory results.