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Applied and Environmental Microbiology, April 2002, p. 1548-1555, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1548-1555.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems

Leonid A. Kulakov,1,2* Morven B. McAlister,4,{dagger} Kimberly L. Ogden,3 Michael J. Larkin,1,2 and John F. O'Hanlon4

The Questor Centre, The Queen's University of Belfast, Belfast BT9 5AG,1 School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland,2 Departments of Chemical and Environmental Engineering,3 Electrical and Computer Engineering, University of Arizona, Tucson, Arizona 857214

Received 7 November 2001/ Accepted 23 January 2002

Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4',6'-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (>=20%). Two sets of primers specific to R. pickettii and Bradyrhizobium sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp. and some P. saccharophilia strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.

* Corresponding author. Mailing address: The Questor Centre, David Keir Building, The Queen's University of Belfast, Belfast BT9 5AG, Northern Ireland. Fax: 44(0)28-90-661462. Phone: 44(0)28-90-274218. E-mail: L.Kulakov{at}qub.ac.uk.

{dagger} Present address: Pall Corporation, Port Washington, NY 11050-4630.

Applied and Environmental Microbiology, April 2002, p. 1548-1555, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1548-1555.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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