Fermentation in 24-well plates is an efficient screening platform for filamentous fungi

Conventional citric acid batch-fermentation by filamentous fungi is often linked with large variations in production levels. The most used citric acid batch-fermentation method described in literature is based on Erlenmeyer flasks with cotton-stoppers in a heated shaker. Our preliminary studies using Erlenmeyer flasks in sextuplicates showed a relative standard sample deviation of 54 % citric acid produced with the filamentous fungi Aspergillus carbonarius, thereby confirming these large variations. The objective of this study was to propose a different and more reliable screening method for citric acid production from filamentous fungi, using A. carbonarius as test organism in a 24 well plate system. The two systems where run simultaneously with only the mechanical parameters as difference between them. All experiments were performed in sextuplicates to be able to give a higher statistical impact than the conventional triplicates. After 5 days fermentation, the citric acid produced in the Erlenmeyer system had a relative standard deviation of 48% compared to the 24-well system that gave a relative standard deviation of 6%. The statistical analysis showed that the difference between the replicates in the 24 well plate system was significantly smaller than in the conventional Erlenmeyer flask setup. This allows for a more precise determination of effects caused by for example changing fermentation parameters or making genetic alterations. We therefore conclude that fermentation in 24-well plates is a more reliable screening method for citric acid production by filamentous fungi, and likely for screening in general. Furthermore by screening in a 24 well plate system, it is possible to screen the same amount of samples as in a conventional flask system, using less space and less materials