engleski

Study of the process development for Escherichia coli based pyruvate production

The commercial demand for pyruvic acid has increased greatly in past decades owing to it various applications, e. g. as an effective starting material for the synthesis of many drugs, agrochemicals and nowadays in food industry as a fat burner. The main goal in this project was the development of a pyruvate production process from glucose with a high molar pyruvate/glucose yield (approaching 2 mol pyruvate/mol glucose) and space-time yield using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate in acetyl-CoA or acetate, resulting in acetate-auxotrophy during growth in glucose minimal medium. Due to the strain genotype, acetate availability was assumed to represent a key fermentation variable. Experimental studies identified a "simple" correlation between acetate consumption rate (ACR) and CO2 production rate (CTR) with an optimum equal molar ratio. Therefore, CTR (calculated on-line by CO2 and O2 exhaust gas analysis) was used for on-line calculation and regulation of the acetate feed (acetate limiting, saturating and accumulating conditions could be established). Glucose closed-loop control was established and series of fed-batch processes were performed. At optimal process conditions final pyruvate titer higher than 700 mmol dm-3 (62 g dm-3), integral molar yields of 1.11 mol pyruvate/mol glucose and a space time yield (STY) of 42 g dm-3 d-1 were achieved. Evidence was obtained that high extra-cellular pyruvate concentration inhibits the process. To face this problem with process engineering means, repetitive fed-batch experiments with cell retention were performed. Molar yield pyruvate/glucose was improved up to 1.7 mol mol-1. STY was increased more than 300 % and has reached 145 g dm-3 h-1. Continuous process with cell retention was developed to simplify complex set-up used for repetative fed-batch process. At optimal process conditions molar yield of 1.24 mol pyruvate/mol glucose and STY higher than 110 g dm-3 d-1 were achieved. To separate pyruvate from fermentation broth fully integrated continuous process has been developed. In this process electro-dialysis was used as a separation unit. Under optimum conditions final (calculated) pyruvate titers higher than 900 mmol dm-3 (79 g dm-3) were achieved. Additionally, unstructured model for the bioconversion of glucose to pyruvate, as well as model of the electrodialysis process were developed, process parameters were estimated and both models were validated.

process development; Escherichia coli; pyruvate; modeling; in situ product recovery

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