Pseudomonas aeruginosa is a highly pathogenic and opportunistic bacterium that is becoming increasingly difficult to treat due to the emergence of antibiotic resistant strains. Previous studies have determined that disturbing the spatial structure of organized bacterial populations can attenuate the expression of virulence factors, including pyoverdine synthesis in P. aeruginosa. Disrupting the spatial structure of organized communities could be developed into a possible treatment that attenuates the expression of virulence factors, including pyoverdine, in the absence of antibiotics. However, it has not yet been determined what the long-term effect of spatial disturbance in various growth environments has on pyoverdine synthesis. To address this open question, we investigated how various metabolites affect the stability of pyoverdine production in disturbed communities composed of P. aeruginosa. We found that bacteria evolved to form denser biofilms capable of producing more pyoverdine, however the evolved bacteria remained susceptible to spatial disturbance.