Analysis of bacteria contaminating ultrapure water in industrial systems

Abstract

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 (>or=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.

FIG. 1.

Schematic presentation of the typical UPW production system studied in this work. Direction of the water flow shown by arrows. Most of the components presented on the diagram are common to all five UPW systems investigated in this work, although the order of some water treatment stages differed. Only UPWS-3 included thermal treatment of the UPW, which was located after the final filters at the beginning of the distribution line (not shown on this diagram). UPWS-2 and UPWS-4 do not have degasification units. Most of the UPW samples (UPWS-2, UPWS-3, UPWS-4, and UPWS-5) analyzed in this work were obtained from the polishing loop, namely, from the ports located at the final stages of UPW production (distribution line; ports located after UV254 treatment and some others). A more detailed survey was completed in the case of UPWS-1. Incoming water samples were used as controls.

FIG. 2.

Phylogenetic analysis of the 16S rRNA genes from strains isolated from University of Arizona UPW System (UPWS-1). The tree was obtained by the neighbor-joining approach by using the TREECON program. Similar phylogenies were obtained when parsimony analysis of the same data was conducted. Bootstrap values (in percentages) are given at the nodes. Bar, 0.02 base substitutions per site. The 16SrRNA gene sequence from Flavobacterium aquatile was used as the outgroup. The GenBank accession numbers of the organisms (in brackets): Roseatales depolymerans DSM11813 (AB003623), Ralstonia (formerly Burkholderia) pickettii MSP3 (AB004790), Pseudomonas syzygii ATCC 49543T (AB021403), Pseudomonas saccharophila DSM654T (AB021407), Matsuebacter chitosanotabidus (AB006851), Ralstonia eutropha DSM2839 (D87999), Bradyrhizobium japonicum USDA94 (D13429), Ralstonia (formerly Pseudomonas) pickettii ATCC 27512 (X67042), Bradyrhizobium elkanii USDA76 (U35000), Sphingomonas sp. strain BF2 (X89905), Bradyrhizobium sp. strain BDV5111 (Z94805), Stenotrophomonas maltophilia LMG 957 (AJ131114), Flavobacterium aquatile ATCC 11947 (M62797), Ralstonia (formerly Burkholderia) solanacearum ACH0732 (U27983), Cytophaga sp. type 0092 (X85210), and Geodermatophilus obscurus DSM43161 (X92355). Accession numbers for strains isolated in the present study (from UPWS-1): 5E (AF368757), MF254A (AF368759), S23 (AF368758), 5F3 (AY039303), 5-1 (AF368755), 3A3C (AF368754), and 3A5 (AF368756).