SPATIAL PHYSIOLOGICAL HETEROGENEITY IN PSEUDOMONAS-AERUGINOSA BIOFILMIS DETERMINED BY OXYGEN AVAILABILITY

The role of oxygen availability in determining the local physiological activity of Pseudomonas aeruginosa growing in biofilms was investigat ed. Biofilms grown in an ambient-air environment expressed approximate ly 1/15th the alkaline phosphatase specific activity of planktonic bac teria subjected to the same phosphate limitation treatment. Biofilms g rown in a gaseous environment of pure oxygen exhibited 1.9 times the a mount of alkaline phosphatase specific activity of air-grown biofilms, whereas biofilms grown in an environment in which the air was replace d with pure nitrogen prior to the inducing treatment did not develop a lkaline phosphatase activity, Frozen cross sections of biofilms staine d for alkaline phosphatase activity with a fluorogenic stain demonstra ted that alkaline phosphatase activity was concentrated in distinct ba nds adjacent to the gaseous interfaces. These bands were approximately 30 mu m thick with biofilms grown in air, 2 mu m thick with biofilms grown in pure nitrogen, and 46 mu m thick with biofilms grown in pure oxygen. Overall biofilm thickness ranged from approximately 117 to app roximately 151 mu m. Measurements with an oxygen microelectrode indica ted that oxygen was depleted locally within the biofilm and that the o xygen-replete zone was of a dimension similar to that of the biologica lly active zone, as indicated by alkaline phosphatase induction, These experiments revealed marked spatial physiological heterogeneity withi n P. aeruginosa biofilms in which active protein synthesis was restric ted by oxygen availability to the upper 30 mu m of the biofilm. Such p hysiological heterogeneity has implications for microbial ecology and for understanding the reduced susceptibilities of biofilms to antimicr obial agents.