APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2002, p. 15481555 0099-2240/02/$04.00 0 DOI: 10.1128/AEM.68.4.15481555.

2002 Copyright 2002, American Society for Microbiology. All Rights Reserved.

Vol. 68, No. 4

Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems

Leonid A. Kulakov,1,2* Morven B. McAlister,4 Kimberly L. Ogden,3 Michael J. Larkin,1,2 and John F. OHanlon4
The Questor Centre, The Queens University of Belfast, Belfast BT9 5AG,1 and School of Biology and Biochemistry, Medical Biology Centre, The Queens University of Belfast, Belfast BT9 7BL,2 Northern Ireland, and Departments of Chemical and Environmental Engineering3 and Electrical and Computer Engineering,4 University of Arizona, Tucson, Arizona 85721
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 epiuorescence microscopy. Assessment of bacterial presence in UPW by epiuorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4 ,6 -diamidino-2-phenylindole] staining) showed signicantly 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 signicant part of the total number of isolated strains (>20%). Two sets of primers specic 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-xing genes in these bacteria. Many industries suffer from the microbial contamination of ultrapure water (UPW). These include the semiconductor, pharmaceutical, food, and beverage industries. Within the semiconductor industry, ultrapure water is utilized in the nal rinsing stage, and the presence of even a single bacterial cell and/or the products of cellular degradation, can severely compromise the quality of the nal product (33, 39). The industrial production of UPW is a complex multistep process, which involves two major stages referred to as pretreatment and polishing (Fig. 1). A variety of steps are included in many UPW production systems (e.g., ltration, UV light treatment, heat treatment, and ozonation) to remove and destroy bacteria. In particular, treatment with UV254 light and ozonation are present in some parts of a facility solely to prevent microbial contamination. Nitrogen gas is often used instead of air above stored UPW to prevent carbon dioxide and oxygen from dissolving in the water. It is imperative that UPW is kept carbon dioxide-free to prevent ionic loading on the mixed-bed ion-exchange resins, while the lowering of oxygen concentration should minimize bacterial growth (Fig. 1). Despite these precautions, piping, membranes, tanks, and other surfaces within the UPW system provide favorable places for bacterial adhesion and cell growth. The complete removal of contaminating microorganisms is considered to be nearly impossible (11, 20). Although UPW contains less than part-per-billion quantities
* Corresponding author. Mailing address: The Questor Centre, David Keir Building, The Queens University of Belfast, Belfast BT9 5AG, Northern Ireland. Fax: 44(0)28-90-661462. Phone: 44(0)28-90274218. E-mail: L.Kulakov@qub.ac.uk. Present address: Pall Corporation, Port Washington, NY 110504630. 1548

of inorganic and organic molecules, a group of microorganisms known as oligotrophs have adapted to these stringent conditions (22, 26). Many of these bacteria can excrete extracellular polysaccharides, allowing both adherence to surfaces and potential resistance to disinfection (14, 16, 37). The extracellular polysaccharide matrix acts as a diffusion barrier to nutrients and cellular products and allows nutrients from the owing water to reach bacterial cells (7). The biolms present in UPW systems may be several cell layers thick (11, 20). The dead cells accumulating in biolms may themselves be used as a carbon source by successive generations of bacteria. This phenomenon is often referred to as cryptic growth (29). The removal or disruption of biolms in piping remains a challenge to UPW users. While investigators have addressed the issue of the microbial contamination of UPW, few studies have been conducted to reveal the diversity of bacterial populations present in UPW. More importantly, except for the work by Pepper et al. (24, 25), most studies have been concerned only with bacterium assessment by agar plating techniques. As outlined previously (4, 18, 19), bacterial enumeration by such methods can lead to a vast underestimation of the actual levels of bacterial presence in various environments. It is generally accepted that gram-negative bacteria predominate in UPW (9, 17, 39), and it was shown that Pseudomonas species can be present in distilling and UPW systems (6, 13, 16). Since high-purity water is widely used in many industries, this manufactured type of environment has acquired global importance. The investigation of bacterial diversity is essential for understanding of microbial populations inhabiting UPW. Such investigations will lead to characterization of the nutritional requirements of UPW bacteria and to the assessment of

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FIG. 1. Schematic presentation of the typical UPW production system studied in this work. Direction of the water ow shown by arrows. Most of the components presented on the diagram are common to all ve 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 nal lters at the beginning of the distribution line (not shown on this diagram). UPWS-2 and UPWS-4 do not have degasication 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 nal 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.

the surfaces used for biolm formation. Identication of the main bacterial groups contaminating UPW may lead to the construction of probes for the detection and real-time monitoring of biocontamination. We investigated here the diversity of bacterial communities in two university and four industrial UPW systems. More emphasis was placed on the microorganisms found in the polishing loops (especially distribution lines) of the systems. Oligonucleotide probes specic to the main bacterial species inhabiting UPW were designed. These probes were then successfully used to directly detect bacteria present in ve different UPW plants. Pseudomonas, Ralstonia, and Bradyrhizobium species were shown to be present in most of the analyzed UPW systems.
MATERIALS AND METHODS Media and bacterial strains. R2A media (28) was used in this work for growth and analysis of the bacterial strains present in UPW. This medium is recommended by American Society for Testing and Materials (ASTM) (1, 2) for testing UPW quality and is therefore widely used in industries. All bacterial strains investigated in this study were isolated from water samples obtained at different UPW plants. Three of the six plants analyzed in this study are used for production of UPW in semiconductor manufacturing processes. Designation of the UPW systems analyzed in this work is as follows: UPWS-1 (University of Arizona experimental UPW system, pilot scale), UPWS-2 (University of Arizona experimental UPW system number 2, bench scale), UPWS-3 (industrial UPW system number 1), UPWS-4 (industrial UPW system number 2), UPWS-5 (industrial UPW system number 3, not semiconductor industry), and UPWS-6 (industrial

UPW system number 3, not semiconductor industry). Figure 1 shows a schematic of the common parts of the analyzed UPW systems. Nitrogen was used in polishing loops of UPWS-1, UPWS-3, UPWS-5, and UPWS-6. UPW sample collection. The procedure for UPW sample collection was described in detail previously (18). Briey, before taking water samples, the ports exteriors were cleaned with 70% ethanol, and water was allowed to ow for 3 min (50 ml/min). Samples were collected into sterile Whirlpak tubes and analyzed within 24 h or sooner depending on the location of the UPW plant. For the epiuorescence microscopy analysis, 10 liters of water was ltered through a black polycarbonate membrane (Nuclepore [Corning], 0.2- m pore size) as described by McAlister et al. (18). For detection of 16S rRNA gene sequences in UPW by PCR (direct PCR) bacterial cells from the UPW were concentrated onto polycarbonate membranes (0.1- m pore size) as described above. The membrane was aseptically removed from the lter holder and transferred to a sterile polypropylene centrifuge tube (50 ml) containing 10 ml of double-lter-sterilized UPW. This was incubated at 25C (180 rpm) overnight. After incubation, the cells were concentrated by centrifugation (8,000 rpm, 15 min) and resuspended in 1 ml of double-ltersterilized UPW. Concentrated water samples were used directly in the PCRs. Epiuorescence microscopy. Cyanotolyl tetrazolium chloride (CTC) and DAPI (4 ,6 -diamidino-2-phenylindole) staining techniques were based on the procedure of Pyle et al. (27). All staining solutions were prepared in UPW and double lter sterilized prior to use. Both CTC and DAPI were obtained from Polysciences (Warrington, Pa.). Stained membranes were examined with an epiuorescence microscope (Olympus BH-2) by using the lter combinations described previously (12). A minimum of 20 microscope elds (using an ocular grid of known dimensions) were counted for each membrane. The DAPI count reected the total number of bacterial cells present, while the CTC count represented the number of cells with the potential for respiration. Determination of 16S rRNA gene sequence. Total DNA was isolated from bacterial cells grown to an optical density at 600 nm of between 0.8 and 1.0. After centrifugation the cells were resuspended in 90 l of 50 mM Tris-HCl buffer (pH

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(5 -GCIWTYTAYGGIAARGGIGG-3 ) and primer 407R (5 -AAICCRCCRC AIACIACRTC-3 ). Prevention of contamination. Because of the very low numbers of bacterial cells present in UPW, possible contamination of samples represents an important issue. For PCR analysis of UPW, the precautions described by Pepper et al. (25) were followed. When bacteria were isolated by plating them on R2A medium, the controls included swabs from the port exterior, autoclaved UPW samples, plating the bacteria present in the surrounding environment, and plating the bacteria from the water entering the UPW system.

8.0) containing sucrose (6.2% [wt/vol]) and EDTA (12 mM). Immediately, 20 l of lysozyme (30 mg/ml; Sigma) was added. After 15 min of incubation at 37C, 15 l of sodium dodecyl sulfate (SDS; 20% [wt/vol]) was added, and the mixture was incubated at 37C for 1 h. The preparation was then extracted with an equal volume of phenol and then with phenol-chloroform (1/1 [vol/vol]). DNA was then precipitated with ethanol, washed once, and nally resuspended in 60 l of Tris-EDTA buffer (30). An almost-complete 16S rRNA gene was amplied by PCR with the following universal primers, described by Pascual et al. (21): forward (5 -AGAGTTTGA TCCTGGCTCAG, positions 8 to 27 [Escherichia coli numbering]) and reverse (5 -AAGGAGGTGATCCAGCCGCA, positions 1541 to 1522). Amplication of the 16S rRNA genes was done with Taq DNA polymerase (Stratagene) in a buffer supplied by the manufacturer. Reactions were carried out in volumes of 25 l with deoxynucleoside triphosphates at 200 M concentrations, primers at 0.15 M each, DNA at 100 to 200 ng, and Taq at 0.5 U per reaction. The following temperature prole was used: denaturation at 95C for 3 min, followed by 30 cycles of 94C for 40 s, 60C for 30 s, and 72C for 1 min. The amplication reactions were carried out by using a Perkin-Elmer DNA Thermal Cycler 480. The PCR products were puried by using GFX PCR DNA and Gel Band Purication Kit (Pharmacia Biotech). When the cloning of PCR fragments was required, Pfu polymerase was used for blunt-end generation, and the resulting products were cloned into the SmaI site of the pUC19 vector plasmid. Plasmid DNA was isolated by standard procedures (30). Puried PCR products or plasmid DNA were used in sequencing reactions with the Taq Dye-Deoxy Terminator Cycle Sequencing Kits (Applied Biosystems and Beckman). The primers used for PCR amplication were also employed for sequencing. Additional sequencing primers were designed on the basis of conserved regions of eubacterial 16S rRNA genes (35), as well as on the basis of preliminary information obtained by sequencing of UPW isolates. The forward primers (E. coli numbering) used in this work were as follows: LK256, 5 -GGT TAAGTCCCGCAACGA-3 (positions 1364 to 1381); LK258, 5 -CTCCTACG GGAGGCAGCA-3 (positions 339 to 356); LK272, 5 -TGCCAGCAGCCGCG GTA-3 (positions 516 to 532); and LK274, 5 -AGCAAACAGGATTAGATAC C-3 (positions 1053 to 1072). The reverse primers were as follows: LK257, 5 -TCGTTGCGGGACTTAACC-3 (positions 1381 to 1364); LK266, 5 -ACTG CTGCCTCCCGTAGGA-3 (positions 358 to 340); LK273, 5 -TACCGCGGCT GCTGGCA-3 (positions 532 to 516); and LK275, 5 -GGCGTGGACTACCA GGGTA-3 (positions 1087 to 1069). The nucleotide sequences of both strands were determined by using automatic sequencers (Applied Biosystems model 373A and the Beckman CEQ 2000 DNA Analysis System). Editing and initial analysis of the sequences was performed by using the DNASIS (Hitachi) software package. Searches for nucleotide and amino acid sequence similarities were done by using the FASTA and BLAST programs (23) and the EMBL and GenBank databases. Alignments of the sequences were performed by using the CLUSTALW program (32). Phylogenetic analysis of the alignment was done by using the PHYLIP (version 3.57c) package (10) and the TREECON program (34). For the PHYLIP analysis, bootstraps were obtained with the SEQBOOT program (100 data sets were generated). Parsimony analyses were done with the DNAPARS programs with ordinary parsimony and randomized input order of the sequences. For the analyses with the TREECON program, Tajima and Nei correction (31) was used, and trees were generated by neighbor joining. PCR detection of bacterial contamination in UPW. Concentrated UPW samples (3 l) were used directly in PCRs essentially as was described by Pepper et al. (25). Conditions for PCR were as described above, except the cycling prole used was as follows: denaturation at 95C for 5 min, followed by 35 cycles of 94C for 40 s, 55C for 30 s, and 72C for 1 min. The preparations were then analyzed by 1.2% agarose gel electrophoresis. For the detection of bacterial contamination in the water samples, three sets of primers were used: universal eubacterial primers (see above), primers designed for Bradyrhizobium sp. (forward primer LK288 [5 -CGTAAAGGGTGCGTAGGCGGGTCTTTA-3 ], positions 509 to 535; reverse primer LK289 [5 -CCCTTTCGGTTAGCGCACCGTCTT-3 , positions 1388 to 1365; the estimated fragment size is 880 bp), and primers designed for Ralstonia pickettii (forward primer LK290 [5 -TGTCCGGAAAGAAATGG CTCTGG-3 ], positions 416 to 438; reverse primer LK291 [5 -CTAACTACTT CTGGTAAAGCCCAC-3 ], positions 1413 to 1390; the estimated fragment size is 975 bp). These sets of primers were designed as specic to bacterial strains found in UPW only and therefore should not be considered species specic (e.g., LK288, apart from Bradyrhizobium sp., is homologous to Nitrobacter spp. and some other bacteria; LK289 is specic only to Bradyrhizobium spp. but not to all Bradyrhizobium strains; the same limitations apply to R. pickettii primers). Detection of nifH genes. To detect the genes responsible for nitrogen xation in UPW bacteria, two previously described primers (38) were used: primer 19F

RESULTS Analysis of the University of Arizona UPW system (UPWS-1) was central to this investigation. Bacterial contamination of this system was regularly monitored for 2.5 years. The results of these surveys have been in part reported elsewhere (18), but no species identication was reported. Subsequently, in a comparative study we analyzed the bacterial communities present in UPWS-1, UPWS-2, UPWS-3, UPWS-4, UPWS-5, and UPWS-6. Isolation and characterization of UPW bacteria. Previous analysis of UPWS-1 showed that the majority of UPW strains are facultative oligotrophs (with no obligatory oligotrophs found). They grew equally well on the full-strength R2A medium and its dilutions (18). R2A media therefore were used in this study. The inuence of incubation times on the enumeration of CFU present in UPWS-1 has been reported previously (18). In accordance with those results, plate counts on R2A medium and bacterial isolations were conducted after 4 weeks of incubation at 25C. All of the isolated strains were puried by using the same media. Preliminary characterization showed that the majority of the strains isolated were gram-negative bacteria. All isolations and bacterial counts were conducted in aerobic conditions as recommended by ASTM (1, 2). Our preliminary experiments also failed to produce bacterial growth under anaerobic conditions (results not shown). After initial characterization of the isolated strains, corresponding 16S rRNA gene sequences were obtained. Sequences of at least 900 bp were determined, and for every group of closely related sequences (homology of 99%), an almost complete 16S rRNA gene sequence (1,400 to 1,500 bp) was obtained for at least one bacterial isolate. The results of phylogenetic analysis of bacteria present in UPW obtained from ve different plants are shown in Table 1, and a phylogenetic tree of the main bacterial strains isolated from UPW (UPWS1) is presented in Fig. 2. All of the UPW samples analyzed were collected from ports situated in the nal (polishing loop) parts of the corresponding UPW systems (mostly the distribution line, indicated in Fig. 1). The results indicate that some bacterial species are present in most or all of the UPW systems analyzed. These bacteria were strains most closely related to R. pickettii (found in four of six analyzed UPW systems), strains related to several Bradyrhizobium sp. (present in all but one of the analyzed UPW systems), and Pseudomonas saccharophilia (found in three UPW systems). These bacterial species constituted a signicant part of the total number of isolated strains ( 20%) (Table 1). It is important to note that Bradyrhizobium strains isolated from UPW required at least 7 days to develop visible colonies on R2A medium (25 to 30C). Under the same incubation conditions, most Ralstonia and Pseudomonas strains grew

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UPW system

Location of sampling ports

Nearest neighbor(s) of main strain in BLAST search of GenBank

No. of isolates (% total)

UPWS-1

Before UV254 (polishing loop)

Ralstonia pickettii Bradyrhizobium sp. Flavobacterium sp. Burkholderia sp. Stenotrophomonas sp. Mycobacterium sp. Bacillus sp. Other Ralstonia pickettii Bradyrhizobium sp. Pseudomonas saccharophilia Other Bradyrhizobium sp. Other Ralstonia pickettii Bradyrhizobium sp. Other Bradyrhizobium sp. Pseudomonas saccharophilia Sphingomonas sp. Other Ralstonia pickettii Pseudomonas uorescens Ralstonia pickettii Pseudomonas saccharophilia Bradyrhizobium sp. Sphingomonas sp. Other

8 (13) 3 (5) 3 (5) 4 (6.7) 5 (8.3) 4 (6.7) 8 (13.3) 25 (41) 8 (24) 12 (36) 4 (12) 9 (27) 6 (60) 4 (40) 4 (66) 1 (17) 1 (17) 4 (25) 4 (25) 4 (25) 4 (25) 6 (100) 6 (28) 5 (24) 1 (5) 2 (10) 3 (14) 4 (19)

UPWS-1

DL

UPWS-2 UPWS-3

After UV254, UV185, and nal lters (0.1 m) DL

UPWS-4

DL and DL (return loop)

UPWS-5 UPWS-6

DL DL, storage tank, before UV254 and after UV254

a Bacterial strains isolated by growth on R2A media were identied on the basis of 16S rRNA gene sequences (see Materials and Methods). The distribution line (DL) is usually after UV254 and UV185 and the nal 0.1- m lters and ultralters.

within 1 to 2 days. Bradyrhizobium strains have not previously been reported in these types of systems. A phylogeny of the typical representatives of these bacterial species is given in Fig. 2. Strains related to R. pickettii and Bradyrhizobium sp. were rst detected in UPWS-1 and continued to appear in every survey conducted on this UPW system. P. saccharophilia strains were isolated from UPWS-1 in February 1999, prior to the plant being sanitized, and have not been found in UPWS-1 since then. This bacterium was also isolated in signicant numbers at UPWS-4 and UPWS-6. None of the above bacterial types were detected in the control samples (i.e., in incoming water or air samples taken on the sites). The other types of bacteria detected mainly in UPWS-1 samples were Stenotrophomonas, Ralstonia, and Flavobacterium spp. This is most likely because this system was analyzed much more completely and repeatedly. Some other bacterial strains were characteristically present in lower numbers or did not appear to be present in more than one UPW system and, in some cases, were also detected in incoming water (e.g., Mycobacterium and Bacillus spp.). Assessment of the extent of bacterial contamination of UPW. In addition to identication of bacterial types described above, bacterial numbers present in the UPW system were determined. We report here the analysis of bacterial contamination in the polishing parts of UPW systems and compare

different UPW systems. UPW samples taken from the ports located at the nal stages of water treatment were analyzed. Bacteria present in UPW were enumerated by both agar plate counts and direct counts by using epiuorescence microscopy. The results of the analysis are presented in Table 2. It is important to note that UPWS-1, UPWS-2, UPWS-3, UPWS-4, and UPWS-5 systems showed approximately the same numbers of bacteria when assessed by plate counts after incubation for 4 weeks (somewhat higher for UPWS-6). Assessment of bacterial presence in UPW by epiuorescence microscopy (CTC and DAPI staining) showed signicantly higher numbers (10 to 100 times more bacterial cells were detected) for UPWS-1, UPWS-2, UPWS-3, UPWS-4, and UPWS-5 systems (Table 2). Only the bacterial numbers obtained for UPWS-5 (by CTC staining) corresponded to those by plate counts. Somewhat higher level of UPWS-6 contamination (as shown by plate counts) may point to the presence of a higher percentage of organics in the water. A signicant proportion of the bacteria present in UPW (50 to 90%) appeared to be composed of nonviable cells. Although the lack of CTC signal does not necessary means that an organism is nonviable, the ratio of DAPI to CTC counts may serve as a preliminary assessment of percentage of nonviable bacteria in the populations. In UPWS-4, the number of nonviable cells is particularly high (Table 2).

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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).

It is noteworthy that bacterial numbers did not vary signicantly in samples obtained from various points of the polishing section of the UPW production systems tested (although a vefold decrease in bacterial numbers may be noted in UPWS-3 after thermal treatment of UPW) (Table 2). It was previously shown that bacteria in UPW systems grow as biolms on the inner surfaces of pipings (11, 20). To conrm the origin of planktonic bacteria investigated in this work, swabs were taken in various parts of the polishing loop (UPWS-1), and bacteria thus collected were identied as de-

scribed above. The analysis of the samples isolated from swabs conrmed the presence of bacterial biolms on the inner surfaces of UPW system. No new genera or species were detected by this analysis (i.e., bacteria isolated showed the same identities as those isolated from UPW samples; Table 1). This analysis indicates that planktonic bacteria species isolated from UPW samples represent true diversity of bacterial populations in UPW systems. Detection of UPW bacteria by PCR analysis. Detection of contaminating bacteria by direct PCR of UPW samples was

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TABLE 2. Assessment of bacterial contamination of UPWa

UPW system and port of sampling Bacterial counts in UPW as determined by: Plating (CFU/ml) CTC staining (viable cells/ml) DAPI staining (cells/ml)

UPWS-1 Before UV185 After UV185 Before UV254 After UV254 After 0.1- m lter DL UPWS-2 Before UV185 After UV185 UPWS-3 DL After UV254 Hot UPWb UPWS-4 (return loop), DL and DL UPWS-5, DL UPWS-6 Before UV254 After UV254 Storage tank

1 1 1 1 8 4 1 1 3 1 2 1 1 1 1

17 16 18 13 10 9 32 21 3 29 2

14 10 5 9 6 8 25 10 2 17 0.5 ND

58 42 38 21 18 10 65 43 10 68 12 ND 123 31 66 25

14 14 6 10 10 8 22 11 2 4 1

6 30 20 20

4 15 20 12

9 8 20 2

8 6 2 2

56 24 6 18

a DL, distribution line; ND, no data obtained. The results are expressed as an average of at least three experiments for plate counts, and duplicate membranes were counted for each sampling port. The UV254 lamp in UPWS-5 was located immediately before nal 0.1- m lters. In the case of UPWS-1, the typical counts are given. b That is, maintained at ca. 80C.

case (ca. 500 bp). The bacterial species identied corresponded to those presented in Table 1, i.e., sequences obtained from the UPWS-1, UPWS-3, and UPWS-5 samples were identied as belonging to R. pickettii and sequences from UPWS-4 were identied as belonging to Sphingomonas sp. (results not shown). Detection of nifH genes in bacteria isolated from UPW. A number of Bradyrhizobium strains were isolated from various analyzed UPW systems. Since nitrogen is used in most of these systems to reduce O2 and CO2, its presence may be instrumental in supporting bacterial growth within UPW systems. Although UPW systems have very low overall level of carbon and organic compounds, locally (cryptic growth in biolms) that level may be sufcient to supply energy for nitrogen xation. As a rst approach to investigate this hypothesis, the distribution of nifH gene sequences in the UPW bacterial community has been analyzed. All bacterial strains isolated from UPWS-1 and a number of isolates obtained from the four other systems were analyzed for the presence of nifH genes. It was shown that nifH gene sequences are present in ca. 60% of Bradyrhizobium strains isolated from UPW. nifH genes were also detected in all four analyzed P. saccharophilia strains. Although the presence of nifH genes in Bradyrhizobium is well documented (38), they are more rarely found among Pseudomonas species (3, 5). No other bacterial isolates analyzed showed the presence of nifH genes. It should be noted that the presence of nifH genes does not necessarily mean the activity of nitrogenase, since this enzyme is regulated at both pre- and posttranslational levels (8). DISCUSSION The bacterial diversity within the UPW systems primarily employed in the semiconductor industry has been investigated. Six UPW systems were analyzed, two smaller university systems and four full-size industrial plants, that were located in

rst reported by Pepper et al. (24, 25). In the present study, we used PCR for the detection of bacterial contamination in different UPW plants employed by the semiconductor and other manufacturers. Two sets of primers specic to R. pickettii and Bradyrhizobium sp., as well as primers universal for eubacteria, were used. The specicity of the primers was tested in control experiments involving various laboratory bacterial strains and the strains isolated from UPW; the identities of PCR products obtained with specic primers were also conrmed by sequencing. The results of direct PCR analysis of UPW are presented in Table 3. These results conrmed the possibility of detection of bacterial contamination of UPW by direct PCR analysis. The results obtained with specic primers correspond to those obtained by identication of the isolated bacteria (Table 1). In some cases there were no PCR products obtained (UPWS-4, After UV254 [see Table 3]), which is probably due to the very low numbers of bacterial cells present in particular UPW samples. Primers specic to R. pickettii and Bradyrhizobium sp. allowed detection of the corresponding bacteria in UPW. It is worth noting that, apart from R. pickettii, the primers designed may target several other species. Although these primers always behaved as specic in our experiments with UPW samples, such a possibility should be taken into account when different UPW systems are analyzed. PCR products obtained with the universal bacterial primers from the UPWS-1, UPWS-3, UPWS-4, and UPWS-5 samples (Table 3) were cloned in the pUC18 vector, and partial sequences of the two or three insertions were obtained in each

TABLE 3. PCR detection of bacteria in UPWa

UPW system (sampling port) Detection of 16S ribosomal DNA sequences: Bacterial (universal primers) Ralstonia pickettii Bradyrhizobium sp.

UPWS-1 DL Before UV254 UPWS-3 DL After UV254 UPWS-4 DL After UV254 UPWS-5 (DL) UPWS-6 After UV254 Storage tank
a Designations of the sampling ports are the same as in Tables 1 and 2. PCR was performed as described in Materials and Methods. , Presence of bacteria in UPW detected by PCR, in most cases sequencing of the corresponding PCR fragments was also performed; , no bacteria detected with the corresponding primers.

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geographically diverse areas of the world. Because the UPW systems varied in size and location, the results obtained are considered to be characteristic for UPW production in general. The samples analyzed here were obtained from the ports located in the polishing sections of UPW systems; hence, the bacterial communities investigated may have a signicant impact on the quality of UPW used in the nal (rinsing) stages of semiconductor production. Five UPW systems showed approximately the same level of bacterial contamination as assessed by different methods. Considering the different locations and sizes of the analyzed UPW systems, the bacteria detected may be considered as indigenous populations typically found in these systems. A comparison of the UPW bacterial contamination by plate counts to that of DAPI and CTC staining detected a signicant underestimation of bacterial presence by the former (with the exception of UPWS-6; similar bacterial numbers were detected by plate counts and epiuorescence microscopy). Similar results have already been reported for drinking water and UPW analysis (4, 18, 19). It is important to note that detection and estimation of bacteria within the semiconductor industry still relies heavily on direct cultivation and plating techniques according to ASTM standards (1, 2). Thus, the results of such procedures may signicantly underestimate the extent of the problem. The bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for UPW production systems. These included R. pickettii, Bradyrhizobium, P. saccharophilia, Stenotrophomonas, and Ralstonia strains. It is worth noting that identication solely on the basis of 16S rRNA gene sequences (this work) is not sufcient for drawing a reliable distinction between species. It is essential to note that UPWS-1 was analyzed far more rigorously than the other four systems; UPW samples were taken from UPWS-1 on a bimonthly basis, and bacteria present were analyzed by plating, epiuorescence microscopy, and PCR. UPW samples from other plants were obtained once or twice during the same period. From the analysis of UPWS-1, it became clear that the above groups of bacteria represent the most important parts of the bacterial community in polishing and distribution parts of the system, whereas various other bacterial species were found in the sections of the plant located upstream of the nal UV lamps and lters (Table 1). Analysis of the other UPW plants showed that the bacterial groups isolated from UPWS-1 were also the main bacteria inhabiting other UPW environments. R. pickettii strains were found in four of the six analyzed UPW systems, and strains identied as mostly close to Bradyrhizobium sp. were present in all but one UPW systems. Previous research showed that various representatives of the genus Pseudomonas are present in UPW (6, 16, 22, 33). P. aeruginosa and Burkholderia cepacia were shown to contaminate a waterdistilling system (13). R. pickettii strains were also reported present among many others species in UPW (6). However, there were no Bradyrhizobium strains detected in UPW previously, and no attempts have been made to identify the typical bacterial strains inhabiting various UPW production systems. Failure to isolate Bradyrhizobium strains from UPW in previous reports may be due to the relatively slower growth of these bacteria on standard R2A media; ASTM guidelines (1, 2)

recommend growing the bacteria (for isolation from UPW) for 48 to 72 h. Our experiments showed that this period of incubation is insufcient for growth of Bradyrhizobium sp. present in UPW systems (18). It is important to emphasize that very little variation in bacterial counts was observed when different parts of UPW polishing loops were analyzed. This observation questions the effectiveness of some stages of antibacterial treatment employed in modern UPW production: in particular, UV254 treatment seems to have little effect on the bacterial numbers present in water. These ndings may be better understood in conjunction with the nature of bacterial growth in UPW systems, which according to most available evidence occurs in the form of biolms attached to inner surfaces (11, 20). If we take into consideration the results of the present study, it may be suggested that each part of the UPW production system (separated from others by lters, UV-units, etc.) possesses its own bacterial population (biolm) relatively independent from others present in the same system. Correspondingly, planktonic bacteria detected in UPW represent cells detached from biolms. Although this suggestion seems reasonable and corresponds with the results obtained, further experimental work may be needed for its conrmation. It is worth noting that Bradyrhizobium strains were detected in all plants where nitrogen had been used; however, the same group of bacteria was also isolated from the UPWS-2, which does not contain nitrogen. The industrial plant seemingly free from Bradyrhizobium sp. did not use nitrogen in its system (UPWS-4). Discovery of nifH genes in Bradyrhizobium strains is not surprising by itself, but when taken in conjunction with the spread of this bacterial group in UPW systems, it may suggest that nitrogen contributes to cell maintenance and growth in these systems. The presence of nifH sequences in P. saccharophilia is somewhat more unusual. However, a few examples of nitrogen-xing Pseudomonas strains have been reported (5, 15, 36), and a nitrogen-xing strain of P. saccharophilia ATCC 15946 has also been reported (3). It is important to stress that nding bacterial strains with nif genes deserves further investigation, since it provides the rst evidence that the widespread use of nitrogen in UPW production systems may contribute to bacterial contamination. Identication of the typical bacterial strains inhabiting UPW systems allowed us to design oligonucleotide primers specic to two main bacterial groups. PCR experiments conducted with UPW samples demonstrated the possibility for detection of R. pickettii and Bradyrhizobium sp. in UPW. A 100- to 1,000fold concentration of water samples was needed for such detection, since bacterial numbers in typical UPW taken from the system were too low to allow direct PCR detection of bacterial 16S rDNA sequences. The use of specic (as well as universal) primers for the detection of the bacterial contamination in UPW systems may be considered useful for the preliminary assessment of bacterial presence in UPW. In conclusion, it should be said that certain bacterial populations appear common to many industrial UPW systems and are represented mostly by gram-negative strains. Several bacterial species were found (Pseudomonas, Ralstonia, and Bradyrhizobium spp.) that seem to be indigenous to an oligotrophic UPW environment.

VOL. 68, 2002 ACKNOWLEDGMENTS

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We acknowledge the support of the NSF Industry/University Cooperative Research Center Program and of the Industrial Research and Technology Unit Northern Ireland START Programme Grant under the international TIE Project, Microbiocontamination in Ultrapure Water, involving researchers at the University of Arizona, The Queens University of Belfast, SUNY at Buffalo, and NJIT. We also thank four industrial sites that allowed us to sample their UPW systems and Jon Sjogren for help with the collection of UPW samples.
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