Table 1.

Optimization of fed-batch cultivation of recombinant PichiaPink™ strains expressing Af Cel12A and Ta Cel5A endoglucanases

Run	Strain	Temperature (°C)	Feed	Feed regime	Max dry cell weight (g/l)	Total supernatant protein (g/l)
1	GAP/TaCel5A	30	Glycerol	1714 g over 70 h	109	2.8
2b	GAP/TaCel5A	30	Glycerol	2470 g over 70 h	155 ± 5	3.8 ± 0.4
3	GAP/TaCel5A	30	Glucose	2470 g over 70 h	145	4.9
4	GAP/TaCel5A	25	Glycerol	2470 g over 70 h	155	5.4
5	GAP/AfCel12A	30	Glycerol	1714 g over 70 h	132	3.4
6b	GAP/AfCel12A	30	Glycerol	2470 g over 70 h	146 ± 9	3.3 ± 0.3
7	GAP/AfCel12A	30	Glucose	2470 g over 70 h	100	n.d.a
8	GAP/AfCel12A	25	Glycerol	2470 g over 70 h	170	7.3

^aNot determined; ^bOptimal parameters, fermentations run in 5 parallels.

Fermentations were carried out to analyze the effect of temperature, increased glycerol feed and carbon source on the GAP/TaCel5A strain and the GAP/AfCel12A strain. The PichiaPink™ cells were grown in 15-l scale bioreactors with a 5-l initial volume for 24 hours in batch mode, followed by feeding as indicated; see Methods for more details. Runs were performed as single experiments; however, optimized runs were performed five times, and standard deviations are provided. SDS-PAGE analyses of the culture supernatants are shown in Figure  3.