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Applied and Environmental Microbiology, October 1998, p. 4035-4039, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Spatial Physiological Heterogeneity in Pseudomonas aeruginosa Biofilm Is Determined by Oxygen Availability

Karen D. Xu,1,2 Philip S. Stewart,1,3,* Fuhu Xia,1 Ching-Tsan Huang,1,dagger and Gordon A. McFeters1,2

Center for Biofilm Engineering,1 Department of Microbiology,2 and Department of Chemical Engineering,3 Montana State University---Bozeman, Bozeman, Montana 59717-3980

Received 23 March 1998/Accepted 20 July 1998

The role of oxygen availability in determining the local physiological activity of Pseudomonas aeruginosa growing in biofilms was investigated. Biofilms grown in an ambient-air environment expressed approximately 1/15th the alkaline phosphatase specific activity of planktonic bacteria subjected to the same phosphate limitation treatment. Biofilms grown in a gaseous environment of pure oxygen exhibited 1.9 times the amount of alkaline phosphatase specific activity of air-grown biofilms, whereas biofilms grown in an environment in which the air was replaced with pure nitrogen prior to the inducing treatment did not develop alkaline phosphatase activity. Frozen cross sections of biofilms stained for alkaline phosphatase activity with a fluorogenic stain demonstrated that alkaline phosphatase activity was concentrated in distinct bands adjacent to the gaseous interfaces. These bands were approximately 30 µm thick with biofilms grown in air, 2 µm thick with biofilms grown in pure nitrogen, and 46 µm thick with biofilms grown in pure oxygen. Overall biofilm thickness ranged from approximately 117 to approximately 151 µm. Measurements with an oxygen microelectrode indicated that oxygen was depleted locally within the biofilm and that the oxygen-replete zone was of a dimension similar to that of the biologically active zone, as indicated by alkaline phosphatase induction. These experiments revealed marked spatial physiological heterogeneity within P. aeruginosa biofilms in which active protein synthesis was restricted by oxygen availability to the upper 30 µm of the biofilm. Such physiological heterogeneity has implications for microbial ecology and for understanding the reduced susceptibilities of biofilms to antimicrobial agents.

* Corresponding author. Mailing address: Department of Chemical Engineering, Montana State University, Bozeman, MT 59717-3980. Phone: (406) 994-2890. Fax: (406) 994-6098. E-mail: phil_s{at}erc.montana.edu.

dagger Present address: Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan, Republic of China.

Applied and Environmental Microbiology, October 1998, p. 4035-4039, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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