Growth experiments were conducted with fusaric acid concentrations ranging from 0 to 250 ug/ml were performed on Eschericia coli (Strains DH5alfa, BL21 and JM109), Bacillus subtilis and Pseudomonas putida. After 18 hours of incubation at 37 degress celcius, OD 600 measurements were taken.

NOTE: After ordering new fusaric acid later in the project, it was found out that the fusaric acid used in this experiment was not the same strength, with the old one most likely being (partially) degraded. Therefore data from this experiment is not used.

Since some soil bacteria are known to be able to degrade fusaric acid (FA), a HPLC experiment was started to test P. putida(PP) for fusaric acid degradation and carbon utilization. M9 media was used with glucose/fusaric acid(250ug/ml) or both as a carbon source.

For the HPLC the following settings were used.

• Column: Polaris C18A
• Eluent: Acetonitril
• Temperature: 35 degrees Celsius
• Flow speed: 0.5 ml/min
• Detection: UV (260 - 280nm)

Based on the initial results a more elaborate experiment was started in duplo. Six samples would be grown in duplo:

• A. Negative control for growth on M9 without carbon source. (PP+, Glucose-, FA-)
• B. Positive control for growth on M9 with carbon source (glucose).(PP+, Glucose+, FA-)
• C. Positive control for growth on M9 with carbon source and fusaric acid. (PP+, Glucose+, FA-)
• D. Test for PP growth on FA as carbon source.(PP+, Glucose-, FA+)
• E. E. Negative control for contamination and FA stability. (PP+, Glucose-, FA-)
• F. Negative control for contamination. (PP-, Glucose+, FA-)

High Performance Liquid Chromatography (HPLC)

Since some soil bacteria are known to be able to degrade fusaric acid (FA), a HPLC experiment was started to test P. putida(PP) for fusaric acid degradation and carbon utilization. M9 media was used with glucose/fusaric acid(250ug/ml) or both as a carbon source.