APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 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† 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 and School of Biology and Biochemistry, Medical
Biology Centre, The Queen’s 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

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and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy
(cyanotolyl tetrazolium chloride [CTC] and DAPI [4ⴕ,6ⴕ-diamidino-2-phenylindole] staining) showed signifi-
cantly 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.

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 final
rinsing stage, and the presence of even a single bacterial cell
and/or the products of cellular degradation, can severely com-
promise the quality of the final product (33, 39).
The industrial production of UPW is a complex multistep
process, which involves two major stages referred to as pre-
treatment and polishing (Fig. 1). A variety of steps are in-
cluded in many UPW production systems (e.g., filtration, 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). De-
spite 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 im-
possible (11, 20).
Although UPW contains less than part-per-billion quantities
* 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@qub.ac.uk.
† Present address: Pall Corporation, Port Washington, NY 11050-
4630.
of inorganic and organic molecules, a group of microorganisms
known as oligotrophs have adapted to these stringent condi-
tions (22, 26). Many of these bacteria can excrete extracellular
polysaccharides, allowing both adherence to surfaces and po-
tential 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 flowing
water to reach bacterial cells (7).
The biofilms present in UPW systems may be several cell
layers thick (11, 20). The dead cells accumulating in biofilms
may themselves be used as a carbon source by successive gen-
erations of bacteria. This phenomenon is often referred to as
cryptic growth (29). The removal or disruption of biofilms 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 assess-
ment 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-neg-
ative 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 nutri-
tional requirements of UPW bacteria and to the assessment of

1548
VOL. 68, 2002 BACTERIA CONTAMINATING ULTRAPURE WATER SYSTEMS 1549

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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.

the surfaces used for biofilm formation. Identification of the
main bacterial groups contaminating UPW may lead to the
construction of probes for the detection and real-time moni-
toring of biocontamination.
We investigated here the diversity of bacterial communities
in two university and four industrial UPW systems. More em-
phasis was placed on the microorganisms found in the polish-
ing loops (especially distribution lines) of the systems. Oligo-
nucleotide probes specific to the main bacterial species
inhabiting UPW were designed. These probes were then suc-
cessfully used to directly detect bacteria present in five differ-
ent 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 recom-
mended 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 produc-
tion 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 exper-
imental 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 de-
scribed in detail previously (18). Briefly, before taking water samples, the ports’
exteriors were cleaned with 70% ethanol, and water was allowed to flow 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 epifluorescence microscopy analysis, 10 liters of water was filtered
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 filter holder and transferred to a sterile polypropylene centrifuge tube
(50 ml) containing 10 ml of double-filter-sterilized UPW. This was incubated at
25°C (180 rpm) overnight. After incubation, the cells were concentrated by
centrifugation (8,000 rpm, 15 min) and resuspended in 1 ml of double-filter-
sterilized UPW. Concentrated water samples were used directly in the PCRs.
Epifluorescence 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 filter sterilized prior to use. Both CTC and DAPI were obtained from
Polysciences (Warrington, Pa.). Stained membranes were examined with an
epifluorescence microscope (Olympus BH-2) by using the filter combinations
described previously (12). A minimum of 20 microscope fields (using an ocular
grid of known dimensions) were counted for each membrane. The DAPI count
reflected the total number of bacterial cells present, while the CTC count rep-
resented 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
1550 KULAKOV ET AL.
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 37°C, 15
␮l of sodium dodecyl sulfate (SDS; 20% [wt/vol]) was added, and the mixture was
incubated at 37°C 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 finally resuspended in 60 ␮l of
Tris-EDTA buffer (30).
An almost-complete 16S rRNA gene was amplified 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). Amplification
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 profile was used: denaturation at 95°C for 3 min, followed by 30
cycles of 94°C for 40 s, 60°C for 30 s, and 72°C for 1 min. The amplification
reactions were carried out by using a Perkin-Elmer DNA Thermal Cycler 480.
The PCR products were purified by using GFX PCR DNA and Gel Band
Purification 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).
Purified 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 amplification were also employed for
sequencing. Additional sequencing primers were designed on the basis of con-
served 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 pro-
gram (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 sam-
ples (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 profile
used was as follows: denaturation at 95°C for 5 min, followed by 35 cycles of 94°C
for 40 s, 55°C for 30 s, and 72°C for 1 min. The preparations were then analyzed
by 1.2% agarose gel electrophoresis. For the detection of bacterial contamina-
tion 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⬘, posi-
tions 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 specific to bacterial strains
found in UPW only and therefore should not be considered species specific (e.g.,
LK288, apart from Bradyrhizobium sp., is homologous to Nitrobacter spp. and
some other bacteria; LK289 is specific 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 fixation
in UPW bacteria, two previously described primers (38) were used: primer 19F
APPL. ENVIRON. MICROBIOL.
(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 impor-
tant 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 me-
dium, the controls included swabs from the port exterior, autoclaved UPW
samples, plating the bacteria present in the surrounding environment, and plat-
ing the bacteria from the water entering the UPW system.
RESULTS
Analysis of the University of Arizona UPW system
(UPWS-1) was central to this investigation. Bacterial contam-
ination of this system was regularly monitored for 2.5 years.
The results of these surveys have been in part reported else-
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where (18), but no species identification was reported. Subse-
quently, in a comparative study we analyzed the bacterial com-
munities 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 me-
dium and its dilutions (18). R2A media therefore were used in
this study. The influence of incubation times on the enumera-
tion 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 25°C. All of the isolated strains were purified
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, corre-
sponding 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 phy-
logenetic analysis of bacteria present in UPW obtained from
five different plants are shown in Table 1, and a phylogenetic
tree of the main bacterial strains isolated from UPW (UPWS-
1) is presented in Fig. 2. All of the UPW samples analyzed
were collected from ports situated in the final (polishing loop)
parts of the corresponding UPW systems (mostly the distribu-
tion 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 Brady-
rhizobium sp. (present in all but one of the analyzed UPW
systems), and Pseudomonas saccharophilia (found in three
UPW systems). These bacterial species constituted a signifi-
cant part of the total number of isolated strains (ⱖ20%) (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 30°C). Under the same incubation
conditions, most Ralstonia and Pseudomonas strains grew
VOL. 68, 2002 BACTERIA CONTAMINATING ULTRAPURE WATER SYSTEMS 1551

TABLE 1. Bacterial strain identification on the basis of 16S rRNA gene analysis in tested UPW systemsa
Nearest neighbor(s) of main strain No. of isolates
UPW system Location of sampling ports
in BLAST search of GenBank (% total)

UPWS-1 Before UV254 (polishing loop) Ralstonia pickettii 8 (13)

Bradyrhizobium sp. 3 (5)
Flavobacterium sp. 3 (5)
Burkholderia sp. 4 (6.7)
Stenotrophomonas sp. 5 (8.3)
Mycobacterium sp. 4 (6.7)
Bacillus sp. 8 (13.3)
Other 25 (41)

UPWS-1 DL Ralstonia pickettii 8 (24)

Bradyrhizobium sp. 12 (36)
Pseudomonas saccharophilia 4 (12)
Other 9 (27)

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UPWS-2 After UV254, UV185, and final Bradyrhizobium sp. 6 (60)
filters (0.1 ␮m) Other 4 (40)

UPWS-3 DL Ralstonia pickettii 4 (66)

Bradyrhizobium sp. 1 (17)
Other 1 (17)

UPWS-4 DL and DL (return loop) Bradyrhizobium sp. 4 (25)

Pseudomonas saccharophilia 4 (25)
Sphingomonas sp. 4 (25)
Other 4 (25)

UPWS-5 DL Ralstonia pickettii 6 (100)

UPWS-6 DL, storage tank, before UV254 Pseudomonas fluorescens 6 (28)

and after UV254 Ralstonia pickettii 5 (24)
Pseudomonas saccharophilia 1 (5)
Bradyrhizobium sp. 2 (10)
Sphingomonas sp. 3 (14)
Other 4 (19)
a
Bacterial strains isolated by growth on R2A media were identified on the basis of 16S rRNA gene sequences (see Materials and Methods). The distribution line
(DL) is usually after UV254 and UV185 and the final 0.1-␮m filters and ultrafilters.

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 first detected in UPWS-1 and contin-
ued to appear in every survey conducted on this UPW system.
P. saccharophilia strains were isolated from UPWS-1 in Feb-
ruary 1999, prior to the plant being sanitized, and have not
been found in UPWS-1 since then. This bacterium was also
isolated in significant numbers at UPWS-4 and UPWS-6. None
of the above bacterial types were detected in the control sam-
ples (i.e., in incoming water or air samples taken on the sites).
The other types of bacteria detected mainly in UPWS-1 sam-
ples 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., Myco-
bacterium and Bacillus spp.).
Assessment of the extent of bacterial contamination of
UPW. In addition to identification of bacterial types described
above, bacterial numbers present in the UPW system were
determined. We report here the analysis of bacterial contam-
ination in the polishing parts of UPW systems and compare
different UPW systems. UPW samples taken from the ports
located at the final stages of water treatment were analyzed.
Bacteria present in UPW were enumerated by both agar plate
counts and direct counts by using epifluorescence 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 num-
bers of bacteria when assessed by plate counts after incubation
for 4 weeks (somewhat higher for UPWS-6). Assessment of
bacterial presence in UPW by epifluorescence microscopy
(CTC and DAPI staining) showed significantly 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 per-
centage of organics in the water. A significant 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 popula-
tions. In UPWS-4, the number of nonviable cells is particularly
high (Table 2).
1552 KULAKOV ET AL. APPL. ENVIRON. MICROBIOL.

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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 signifi-
cantly in samples obtained from various points of the polishing
section of the UPW production systems tested (although a
fivefold 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 biofilms on the inner surfaces of pipings (11, 20). To confirm
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 identified as de-
scribed above. The analysis of the samples isolated from swabs
confirmed the presence of bacterial biofilms on the inner sur-
faces of UPW system. No new genera or species were detected
by this analysis (i.e., bacteria isolated showed the same iden-
tities 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
VOL. 68, 2002 BACTERIA CONTAMINATING ULTRAPURE WATER SYSTEMS 1553

TABLE 2. Assessment of bacterial contamination of UPWa case (ca. 500 bp). The bacterial species identified corresponded
Bacterial counts in UPW as determined by:
to those presented in Table 1, i.e., sequences obtained from the
UPW system and UPWS-1, UPWS-3, and UPWS-5 samples were identified
port of sampling Plating CTC staining DAPI staining
(CFU/ml) (viable cells/ml) (cells/ml)
as belonging to R. pickettii and sequences from UPWS-4
were identified as belonging to Sphingomonas sp. (results not
UPWS-1 shown).
Before UV185 1⫾1 17 ⫾ 14 58 ⫾ 14
Detection of nifH genes in bacteria isolated from UPW. A
After UV185 ⬍1 16 ⫾ 10 42 ⫾ 14
Before UV254 ⬍1 18 ⫾ 5 38 ⫾ 6 number of Bradyrhizobium strains were isolated from various
After UV254 ⬍1 13 ⫾ 9 21 ⫾ 10 analyzed UPW systems. Since nitrogen is used in most of these
After 0.1-␮m filter 8⫾4 10 ⫾ 6 18 ⫾ 10 systems to reduce O2 and CO2, its presence may be instrumen-
DL ⬍1 9⫾8 10 ⫾ 8 tal in supporting bacterial growth within UPW systems. Al-
UPWS-2 though UPW systems have very low overall level of carbon and
Before UV185 1⫾1 32 ⫾ 25 65 ⫾ 22 organic compounds, locally (cryptic growth in biofilms) that
After UV185 3⫾2 21 ⫾ 10 43 ⫾ 11 level may be sufficient to supply energy for nitrogen fixation.
As a first approach to investigate this hypothesis, the distribu-

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UPWS-3
DL ⬍1 3⫾2 10 ⫾ 2 tion of nifH gene sequences in the UPW bacterial community
After UV254 ⬍1 29 ⫾ 17 68 ⫾ 4 has been analyzed. All bacterial strains isolated from UPWS-1
Hot UPWb ⬍1 2 ⫾ 0.5 12 ⫾ 1 and a number of isolates obtained from the four other systems
UPWS-4 (return loop), ⬍1 ND ND were analyzed for the presence of nifH genes. It was shown that
DL and DL nifH gene sequences are present in ca. 60% of Bradyrhizobium
strains isolated from UPW. nifH genes were also detected in all
UPWS-5, DL 6⫾4 9⫾8 123 ⫾ 56
four analyzed P. saccharophilia strains. Although the presence
UPWS-6 of nifH genes in Bradyrhizobium is well documented (38), they
Before UV254 30 ⫾ 15 8⫾6 31 ⫾ 24 are more rarely found among Pseudomonas species (3, 5). No
After UV254 20 ⫾ 20 20 ⫾ 2 66 ⫾ 6
Storage tank 20 ⫾ 12 2⫾2 25 ⫾ 18 other bacterial isolates analyzed showed the presence of nifH
genes. It should be noted that the presence of nifH genes does
a
DL, distribution line; ND, no data obtained. The results are expressed as an not necessarily mean the activity of nitrogenase, since this en-
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 zyme is regulated at both pre- and posttranslational levels (8).
immediately before final 0.1-␮m filters. In the case of UPWS-1, the typical counts
are given.
b
That is, maintained at ca. 80°C. DISCUSSION

The bacterial diversity within the UPW systems primarily

first reported by Pepper et al. (24, 25). In the present study, we employed in the semiconductor industry has been investigated.
used PCR for the detection of bacterial contamination in dif- Six UPW systems were analyzed, two smaller university sys-
ferent UPW plants employed by the semiconductor and other tems and four full-size industrial plants, that were located in
manufacturers. Two sets of primers specific to R. pickettii and
Bradyrhizobium sp., as well as primers universal for eubacteria,
were used. The specificity of the primers was tested in control
TABLE 3. PCR detection of bacteria in UPWa
experiments involving various laboratory bacterial strains and
the strains isolated from UPW; the identities of PCR products Detection of 16S ribosomal DNA sequences:
UPW system
obtained with specific primers were also confirmed by sequenc- (sampling port) Bacterial Ralstonia Bradyrhizobium
ing. The results of direct PCR analysis of UPW are presented (universal primers) pickettii sp.
in Table 3. These results confirmed the possibility of detection UPWS-1
of bacterial contamination of UPW by direct PCR analysis. DL ⫹ ⫹ ⫹
The results obtained with specific primers correspond to those Before UV254 ⫹ ⫹ ⫹
obtained by identification of the isolated bacteria (Table 1). In
UPWS-3
some cases there were no PCR products obtained (UPWS-4, DL ⫹ ⫹ ⫺
“After UV254” [see Table 3]), which is probably due to the After UV254 ⫹ ⫹ ⫺
very low numbers of bacterial cells present in particular UPW
UPWS-4
samples. Primers specific to R. pickettii and Bradyrhizobium sp. DL ⫹ ⫺ ⫹
allowed detection of the corresponding bacteria in UPW. It is After UV254 ⫺ ⫺ ⫺
worth noting that, apart from R. pickettii, the primers designed
may target several other species. Although these primers al- UPWS-5 (DL) ⫹ ⫹ ⫺
ways behaved as specific in our experiments with UPW sam- UPWS-6
ples, such a possibility should be taken into account when After UV254 ⫹ ⫹ ⫺
different UPW systems are analyzed. Storage tank ⫹ ⫹ ⫹
PCR products obtained with the universal bacterial primers a
Designations of the sampling ports are the same as in Tables 1 and 2. PCR
from the UPWS-1, UPWS-3, UPWS-4, and UPWS-5 samples was performed as described in Materials and Methods. ⫹, Presence of bacteria
in UPW detected by PCR, in most cases sequencing of the corresponding PCR
(Table 3) were cloned in the pUC18 vector, and partial se- fragments was also performed; ⫺, no bacteria detected with the corresponding
quences of the two or three insertions were obtained in each primers.
1554 KULAKOV ET AL.
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 lo-
cated in the polishing sections of UPW systems; hence, the
bacterial communities investigated may have a significant im-
pact on the quality of UPW used in the final (rinsing) stages of
semiconductor production.
Five UPW systems showed approximately the same level of
bacterial contamination as assessed by different methods. Con-
sidering 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 significant underestimation
of bacterial presence by the former (with the exception of
UPWS-6; similar bacterial numbers were detected by plate
counts and epifluorescence 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 accord-
ing to ASTM standards (1, 2). Thus, the results of such pro-
cedures may significantly underestimate the extent of the prob-
lem.
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, Brady-
rhizobium, P. saccharophilia, Stenotrophomonas, and Ralstonia
strains. It is worth noting that identification solely on the basis
of 16S rRNA gene sequences (this work) is not sufficient 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, epifluorescence 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 final UV
lamps and filters (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 identified as mostly close to Bradyrhizo-
bium 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 water-
distilling 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 previ-
ously, 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 previ-
ous reports may be due to the relatively slower growth of these
bacteria on standard R2A media; ASTM guidelines (1, 2)
APPL. ENVIRON. MICROBIOL.
recommend growing the bacteria (for isolation from UPW) for
48 to 72 h. Our experiments showed that this period of incu-
bation is insufficient 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 em-
ployed in modern UPW production: in particular, UV254
treatment seems to have little effect on the bacterial numbers
present in water. These findings may be better understood in
conjunction with the nature of bacterial growth in UPW sys-
tems, which according to most available evidence occurs in the
form of biofilms attached to inner surfaces (11, 20). If we take
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into consideration the results of the present study, it may be
suggested that each part of the UPW production system (sep-
arated from others by filters, UV-units, etc.) possesses its own
bacterial population (biofilm) relatively independent from oth-
ers present in the same system. Correspondingly, planktonic
bacteria detected in UPW represent cells detached from bio-
films. Although this suggestion seems reasonable and corre-
sponds with the results obtained, further experimental work
may be needed for its confirmation. It is worth noting that
Bradyrhizobium strains were detected in all plants where nitro-
gen had been used; however, the same group of bacteria was
also isolated from the UPWS-2, which does not contain nitro-
gen. 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. saccharo-
philia is somewhat more unusual. However, a few examples of
nitrogen-fixing Pseudomonas strains have been reported (5, 15,
36), and a nitrogen-fixing strain of P. saccharophilia ATCC
15946 has also been reported (3). It is important to stress that
finding bacterial strains with nif genes deserves further inves-
tigation, since it provides the first evidence that the widespread
use of nitrogen in UPW production systems may contribute to
bacterial contamination.
Identification of the typical bacterial strains inhabiting UPW
systems allowed us to design oligonucleotide primers specific
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 1,000-
fold concentration of water samples was needed for such de-
tection, 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 specific (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 popu-
lations appear common to many industrial UPW systems and
are represented mostly by gram-negative strains. Several bac-
terial species were found (Pseudomonas, Ralstonia, and Brady-
rhizobium spp.) that seem to be indigenous to an oligotrophic
UPW environment.
VOL. 68, 2002 BACTERIA CONTAMINATING ULTRAPURE WATER SYSTEMS
ACKNOWLEDGMENTS
We acknowledge the support of the NSF Industry/University Coop-
erative Research Center Program and of the Industrial Research and
Technology Unit Northern Ireland START Programme Grant un-
der the international “TIE” Project, “Microbiocontamination in Ultra-
pure Water,” involving researchers at the University of Arizona, The
Queen’s 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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