Date

2014

Author

Öztürk, Sibel

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In this study, the aim was to develop an efficient semi-defined medium based continuous feeding strategy for recombinant human growth hormone (rhGH) production by Bacillus subtilis 1A178 scoC knockout (scoC-) strain carrying pMK4::pre(subC)::hGH plasmid. In this context, firstly laboratory-scale air-filtered shake bioreactor experiments were carried out in order to investigate the effects of peptone as the complex carbon and nitrogen source on the cell growth and rhGH production both in the absence and the presence of MgSO4. The highest cell and rhGH concentrations were achieved as CX=1.92 g L-1 and CrhGH=140 mg L-1, respectively, at t=24 h in the semi-defined medium PM-8, containing 8 g L-1 peptone and 0.122 g L-1 MgSO4 in addition to the defined production medium consisting of 8 g L-1 glucose, 4.71 g L-1 (NH4)2HPO4, 2 g L-1 KH2PO4, 0.043 g L-1 Na2HPO4, 5.63 g L-1 NaH2PO4, and 5 ml L-1 trace salt solution (PTM1). Based on these results, in the second phase of the research, MgSO4-containing production medium was used for pilot-scale bioreactor experiments together with the feed substrate stock solutions containing peptone at two different concentrations. Six continuous feeding strategies were designed for semi-batch rhGH production process based on the pre-determined specific growth rates, μ0=0.10, 0.17 and 0.25 h-1, and the transition cultivation times from batch to semi-batch operation, tT=2 h and tT=4 h, were investigated. The highest rhGH concentration was obtained as CrhGH=497 mg L-1 in the semi-defined medium based feeding strategy conducted at μ0=0.25 h-1 where the transition cultivation time was tT=4 h by using the feed substrate stock solution comprising of 200 g L-1 glucose, 117.65 g L-1 (NH4)2HPO4, 100 g L-1 peptone and 5 mL L-1 PTM1 at t=22 h when the cell concentration was reached to CX=8.29 g L-1. The overall product and cell yield on total substrate were attained as Y'(P/S)=7.21 g kg-1 and Y'(X/S)=0.12 g g-1, respectively.

Subject Keywords

Somatotropin., Bacillus subtilis., Bacillus (Bacteria)., Recombinant human somatotropin.

URI

http://etd.lib.metu.edu.tr/upload/12617643/index.pdf
https://hdl.handle.net/11511/23777

Collections

Graduate School of Natural and Applied Sciences, Thesis