Monday, November 5, 2007 - 4:20 PM

In Situ Product Removal As An Approach To Improve The Fermentative Production Of Clavulanic Acid

Simone Brethauer, Martin Held, and Sven Panke. Institute of Process Engineering, ETH Zurich, Universitaetsstrasse 6, Zurich, Switzerland

Clavulanic acid (CA) is a potent β-lactamase inhibitor, which is used clinically in combination with conventional β-lactam antibiotics to treat infections caused by bacteria that would otherwise be resistant to these antibiotics. It is produced on large scale by fermentation of S. clavuligerus, however yields are hampered by the simultaneous decomposition of the product. One generic method to prevent the product loss during fermentation is to apply an in situ product removal (ISPR) strategy, thereby removing the compound of interest from the vicinity of the cells into a stabilizing environment. In the presented work the potential benefits of ISPR are analyzed with respect to the prevention of product decomposition, but also with respect to inhibitory effects caused by the product or its decomposition products. To enable the investigation of product inhibitory effects, first a 12-fold parallelized cultivation device was implemented, which allows the continuous measurement of the oxygen transfer rate (OTR), a key parameter for every aerobic culture, based on a respirometric principle. After proving the validity of the applied principle employing E. coli cultures growing on defined medium, further optimization steps finally allowed to culture S. clavuligerus on a 50 mL scale under conditions and with yields comparable to those of an aerated stirred tank reactor. Employing this device, it was shown, that increased levels of CA severely interfered with the physiology of the producing strain at least at and above 1.5 g L-1 CA, as indicated by a dose-dependent decrease in maximum OTR. However, inhibition of CA synthesis at the level of the current maximum wild-type titer of 0.4 g L-1 could not be observed, as CA still continued to accumulate in the medium after addition of CA in this concentration range. Further investigations elucidated that CA itself and not its decomposition products are responsible for the observed drastic effects as the decay products exert a much less severe effect on growing S. clavuligerus cultures. Finally, ion exchange adsorption was examined as a possible method for ISPR. CA could be immobilized effectively on Diaion TSA1200, but contact times had to be kept short because CA has only limited stability on the resin as well. Thus an ISPR protocol was developed in which CA was removed batchwise from the fermentation broth by treating it twice a day for a limited time with Diaion TSA1200. By this, a total CA concentration of 0.8 g L-1 could be reached compared to 0.4 g L-1 in the control culture without ISPR. To compensate for nutrient removal, a soybean extract was intermittently fed and allowed the net formation of CA to further increase to 1.7 g L-1, which was 3 times higher than in the corresponding control culture representing a clear motivation to pursue ISPR as a technology for yield increase in the CA production process.