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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2002, p. 15481555 Vol. 68, No. 40099-2240/02/$04.000 DOI: 10.1128/AEM.68.4.15481555.2002Copyright 2002, American Society for Microbiology. All Rights Reserved.

Analysis of Bacteria Contaminating Ultrapure Water in Industrial SystemsLeonid 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, MedicalBiology 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 UPWsystems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and otherindustries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate countsand epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy(cyanotolyl tetrazolium chloride [CTC] and DAPI [4,6-diamidino-2-phenylindole] staining) showed signifi-cantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by platecounts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give apositive 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 asignificant 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 theconcentrated 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 associatedwith the presence of nitrogen-fixing genes in these bacteria.

Many industries suffer from the microbial contamination ofultrapure water (UPW). These include the semiconductor,pharmaceutical, food, and beverage industries. Within thesemiconductor industry, ultrapure water is utilized in the finalrinsing stage, and the presence of even a single bacterial celland/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 multistepprocess, 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, UVlight treatment, heat treatment, and ozonation) to remove anddestroy bacteria. In particular, treatment with UV254 light andozonation are present in some parts of a facility solely toprevent microbial contamination. Nitrogen gas is often usedinstead of air above stored UPW to prevent carbon dioxide andoxygen from dissolving in the water. It is imperative that UPWis kept carbon dioxide-free to prevent ionic loading on themixed-bed ion-exchange resins, while the lowering of oxygen

concentration should minimize bacterial growth (Fig. 1). De-spite these precautions, piping, membranes, tanks, and othersurfaces within the UPW system provide favorable places forbacterial adhesion and cell growth. The complete removal ofcontaminating microorganisms is considered to be nearly im-possible (11, 20).

Although UPW contains less than part-per-billion quantities

of inorganic and organic molecules, a group of microorganismsknown as oligotrophs have adapted to these stringent condi-tions (22, 26). Many of these bacteria can excrete extracellularpolysaccharides, allowing both adherence to surfaces and po-tential resistance to disinfection (14, 16, 37). The extracellularpolysaccharide matrix acts as a diffusion barrier to nutrientsand cellular products and allows nutrients from the flowingwater to reach bacterial cells (7).

The biofilms present in UPW systems may be several celllayers thick (11, 20). The dead cells accumulating in biofilmsmay themselves be used as a carbon source by successive gen-erations of bacteria. This phenomenon is often referred to ascryptic growth (29). The removal or disruption of biofilms inpiping remains a challenge to UPW users.

While investigators have addressed the issue of the microbialcontamination of UPW, few studies have been conducted toreveal 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 vastunderestimation of the actual levels of bacterial presence invarious environments. It is generally accepted that gram-neg-ative bacteria predominate in UPW (9, 17, 39), and it wasshown that Pseudomonas species can be present in distillingand UPW systems (6, 13, 16).

Since high-purity water is widely used in many industries,this manufactured type of environment has acquired globalimportance. The investigation of bacterial diversity is essentialfor 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

* Corresponding author. Mailing address: The Questor Centre,David Keir Building, The Queens University of Belfast, Belfast BT95AG, Northern Ireland. Fax: 44(0)28-90-661462. Phone: 44(0)28-90-274218. E-mail: [email protected].

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

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the surfaces used for biofilm formation. Identification of themain bacterial groups contaminating UPW may lead to theconstruction of probes for the detection and real-time moni-toring of biocontamination.

We investigated here the diversity of bacterial communitiesin 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 speciesinhabiting UPW were designed. These probes were then suc-cessfully used to directly detect bacteria present in five differ-ent UPW plants. Pseudomonas, Ralstonia, and Bradyrhizobiumspecies 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

25C (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

FIG. 1. Schematic presentation of the typical UPW production system studied in this work. Direction of the water flow shown by arrows. Mostof the components presented on the diagram are common to all five UPW systems investigated in this work, although the order of some watertreatment stages differed. Only UPWS-3 included thermal treatment of the UPW, which was located after the final filters at the beginning of thedistribution 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 stagesof UPW production (distribution line; ports located after UV254 treatment and some others). A more detailed survey was completed in the caseof UPWS-1. Incoming water samples were used as controls.

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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 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-AGAGTTTGATCCTGGCTCAG, 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 95C for 3 min, followed by 30

cycles of 94C for 40 s, 60C for 30 s, and 72C 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 Biosystemsand 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 theDNASIS (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 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 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 speci fic 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

(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-

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 strainsare facultative oligotrophs (with no obligatory oligotrophsfound). They grew equally well on the full-strength R2A me-dium and its dilutions (18). R2A media therefore were used inthis 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 R2Amedium and bacterial isolations were conducted after 4 weeksof incubation at 25C. All of the isolated strains were purifiedby using the same media. Preliminary characterization showedthat the majority of the strains isolated were gram-negativebacteria. All isolations and bacterial counts were conductedin aerobic conditions as recommended by ASTM (1, 2). Our

preliminary experiments also failed to produce bacterialgrowth under anaerobic conditions (results not shown).

After initial characterization of the isolated strains, corre-sponding 16S rRNA gene sequences were obtained. Sequencesof at least 900 bp were determined, and for every group ofclosely related sequences (homology of 99%), an almostcomplete 16S rRNA gene sequence (1,400 to 1,500 bp) wasobtained for at least one bacterial isolate. The results of phy-logenetic analysis of bacteria present in UPW obtained fromfive different plants are shown in Table 1, and a phylogenetictree 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 bacteriawere strains most closely related to R. pickettii (found in four ofsix analyzed UPW systems), strains related to several Brady-rhizobium sp. (present in all but one of the analyzed UPWsystems), and Pseudomonas saccharophilia (found in threeUPW systems). These bacterial species constituted a signifi-cant part of the total number of isolated strains (20%) (Table1). It is important to note that Bradyrhizobium strains isolatedfrom UPW required at least 7 days to develop visible colonieson R2A medium (25 to 30C). Under the same incubationconditions, most Ralstonia and Pseudomonas strains grew

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within 1 to 2 days. Bradyrhizobium strains have not previouslybeen reported in these types of systems.

A phylogeny of the typical representatives of these bacterialspecies is given in Fig. 2. Strains related to R. pickettii andBradyrhizobium 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 notbeen found in UPWS-1 since then. This bacterium was alsoisolated in significant numbers at UPWS-4 and UPWS-6. Noneof 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 Flavobacteriumspp. This is most likely because this system was analyzed muchmore completely and repeatedly. Some other bacterial strains

were characteristically present in lower numbers or did notappear to be present in more than one UPW system and, insome 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 describedabove, bacterial numbers present in the UPW system weredetermined. 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 portslocated at the final stages of water treatment were analyzed.Bacteria present in UPW were enumerated by both agar platecounts and direct counts by using epifluorescence microscopy.The results of the analysis are presented in Table 2. It isimportant 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 incubationfor 4 weeks (somewhat higher for UPWS-6). Assessment ofbacterial presence in UPW by epifluorescence microscopy(CTC and DAPI staining) showed significantly higher numbers

(10 to 100 times more bacterial cells were detected) forUPWS-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 shownby plate counts) may point to the presence of a higher per-centage of organics in the water. A significant proportion ofthe bacteria present in UPW (50 to 90%) appeared to becomposed of nonviable cells. Although the lack of CTC signaldoes not necessary means that an organism is nonviable, theratio of DAPI to CTC counts may serve as a preliminaryassessment of percentage of nonviable bacteria in the popula-tions. In UPWS-4, the number of nonviable cells is particularlyhigh (Table 2).

TABLE 1. Bacterial strain identification on the basis of 16S rRNA gene analysis in tested UPW systemsa

UPW system Location of sampling portsNearest neighbor(s) of main strain

in BLAST search of GenBankNo. of isolates

(% 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)

UPWS-2 After UV254, UV185, and finalfilters (0.1 m)

Bradyrhizobium sp. 6 (60)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 UV254and after UV254

Pseudomonas fluorescens 6 (28) 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 identi fied 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.


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It is noteworthy that bacterial numbers did not vary signifi-cantly in samples obtained from various points of the polishingsection of the UPW production systems tested (although afi vefold decrease in bacterial numbers may be noted inUPWS-3 after thermal treatment of UPW) (Table 2).

It was previously shown that bacteria in UPW systems growas biofilms on the inner surfaces of pipings (11, 20). To confirmthe 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 swabsconfirmed the presence of bacterial biofilms on the inner sur-faces of UPW system. No new genera or species were detectedby this analysis (i.e., bacteria isolated showed the same iden-tities as those isolated from UPW samples; Table 1). Thisanalysis indicates that planktonic bacteria species isolated fromUPW samples represent true diversity of bacterial populationsin UPW systems.

Detection of UPW bacteria by PCR analysis. Detection ofcontaminating bacteria by direct PCR of UPW samples was

FIG. 2. Phylogenetic analysis of the 16S rRNA genes from strains isolated from University of Arizona UPW System (UPWS-1). The tree wasobtained by the neighbor-joining approach by using the TREECON program. Similar phylogenies were obtained when parsimony analysis of thesame data was conducted. Bootstrap values (in percentages) are given at the nodes. Bar, 0.02 base substitutions per site. The 16SrRNA genesequence 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 maltophiliaLMG 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).


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first reported by Pepper et al. (24, 25). In the present study, we

used PCR for the detection of bacterial contamination in dif-ferent UPW plants employed by the semiconductor and othermanufacturers. Two sets of primers specific to R. pickettii andBradyrhizobium sp., as well as primers universal for eubacteria,were used. The specificity of the primers was tested in controlexperiments involving various laboratory bacterial strains andthe strains isolated from UPW; the identities of PCR productsobtained with specific primers were also confirmed by sequenc-ing. The results of direct PCR analysis of UPW are presentedin Table 3. These results confirmed the possibility of detectionof bacterial contamination of UPW by direct PCR analysis.The results obtained with specific primers correspond to thoseobtained by identification 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 thevery low numbers of bacterial cells present in particular UPWsamples. Primers specific 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 designedmay target several other species. Although these primers al-

ways behaved as specific in our experiments with UPW sam-ples, such a possibility should be taken into account whendifferent UPW systems are analyzed.

PCR products obtained with the universal bacterial primersfrom the UPWS-1, UPWS-3, UPWS-4, and UPWS-5 samples(Table 3) were cloned in the pUC18 vector, and partial se-quences of the two or three insertions were obtained in each

case (ca. 500 bp). The bacterial species identified correspondedto those presented in Table 1, i.e., sequences obtained from theUPWS-1, UPWS-3, and UPWS-5 samples were identifiedas belonging to R. pickettii and sequences from UPWS-4

were identified as belonging to Sphingomonas sp. (results notshown).

Detection of nifH genes in bacteria isolated from UPW. Anumber of Bradyrhizobium strains were isolated from variousanalyzed UPW systems. Since nitrogen is used in most of thesesystems to reduce O

2and CO

2, its presence may be instrumen-

tal in supporting bacterial growth within UPW systems. Al-though UPW systems have very low overall level of carbon andorganic compounds, locally (cryptic growth in biofilms) thatlevel may be sufficient to supply energy for nitrogen fixation.

As a first approach to investigate this hypothesis, the distribu-tion ofnifHgene sequences in the UPW bacterial communityhas been analyzed. All bacterial strains isolated from UPWS-1and a number of isolates obtained from the four other systems

were analyzed for the presence ofnifHgenes. It was shown thatnifHgene sequences are present in ca. 60% of Bradyrhizobium

strains isolated from UPW. nifHgenes were also detected in allfour analyzed P. saccharophilia strains. Although the presenceofnifHgenes in Bradyrhizobium is well documented (38), theyare more rarely found among Pseudomonas species (3, 5). Noother bacterial isolates analyzed showed the presence of nifHgenes. It should be noted that the presence ofnifHgenes doesnot necessarily mean the activity of nitrogenase, since this en-zyme is regulated at both pre- and posttranslational levels (8).

DISCUSSION

The bacterial diversity within the UPW systems primarilyemployed in the semiconductor industry has been investigated.

Six UPW systems were analyzed, two smaller university sys-tems and four full-size industrial plants, that were located in

TABLE 2. Assessment of bacterial contamination of UPWa

UPW system andport of sampling

Bacterial counts in UPW as determined by:

Plating(CFU/ml)

CTC staining(viable cells/ml)

DAPI staining(cells/ml)

UPWS-1Before UV185 1 1 17 14 58 14

After UV185 1 16 10 42 14Before UV254 1 18 5 38 6After UV254 1 13 9 21 10After 0.1-m filter 8 4 10 6 18 10DL 1 9 8 10 8

UPWS-2Before UV185 1 1 32 25 65 22 After UV185 3 2 21 10 43 11

UPWS-3DL 1 3 2 10 2After UV254 1 29 17 68 4Hot UPWb 1 2 0.5 12 1

UPWS-4 (return loop),DL and DL

1 ND ND

UPWS-5, DL 6 4 9 8 123 56

UPWS-6Before UV254 30 15 8 6 31 24 After UV254 20 20 20 2 66 6Storage tank 20 12 2 2 25 18

a DL, distribution line; ND, no data obtained. The results are expressed as anaverage of at least three experiments for plate counts, and duplicate membranes

were counted for each sampling port. The UV254 lamp in UPWS-5 was locatedimmediately before final 0.1-m filters. In the case of UPWS-1, the typical countsare given.

b That is, maintained at ca. 80C.

TABLE 3. PCR detection of bacteria in UPWa

UPW system(sampling port)

Detection of 16S ribosomal DNA sequences:

Bacterial(universal primers)

Ralstoniapickettii

Bradyrhizobiumsp.

UPWS-1DL Before UV254

UPWS-3

DL After UV254

UPWS-4DL After UV254

UPWS-5 (DL)

UPWS-6After UV254 Storage tank

a Designations of the sampling ports are the same as in Tables 1 and 2. PCRwas performed as described in Materials and Methods. , Presence of bacteriain UPW detected by PCR, in most cases sequencing of the corresponding PCRfragments was also performed; , no bacteria detected with the correspondingprimers.


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geographically diverse areas of the world. Because the UPWsystems varied in size and location, the results obtained areconsidered 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, thebacterial communities investigated may have a significant im-

pact on the quality of UPW used in the final (rinsing) stages ofsemiconductor 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 UPWsystems, the bacteria detected may be considered as indigenouspopulations typically found in these systems. A comparison ofthe UPW bacterial contamination by plate counts to that ofDAPI and CTC staining detected a significant underestimationof bacterial presence by the former (with the exception ofUPWS-6; similar bacterial numbers were detected by platecounts and epifluorescence microscopy). Similar results havealready 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 reliesheavily 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 mostlygram negative, and several groups seem to be indigenous forUPW production systems. These included R. pickettii, Brady-rhizobium, P. saccharophilia, Stenotrophomonas, and Ralstoniastrains. It is worth noting that identification solely on the basisof 16S rRNA gene sequences (this work) is not sufficient fordrawing a reliable distinction between species. It is essential tonote that UPWS-1 was analyzed far more rigorously than the

other four systems; UPW samples were taken from UPWS-1on a bimonthly basis, and bacteria present were analyzed byplating, epifluorescence microscopy, and PCR. UPW samplesfrom other plants were obtained once or twice during the sameperiod.

From the analysis of UPWS-1, it became clear that the abovegroups of bacteria represent the most important parts of thebacterial community in polishing and distribution parts of thesystem, whereas various other bacterial species were found inthe sections of the plant located upstream of the final UVlamps and filters (Table 1). Analysis of the other UPW plantsshowed that the bacterial groups isolated from UPWS-1 werealso the main bacteria inhabiting other UPW environments. R.

pickettii strains were found in four of the six analyzed UPWsystems, and strains identified as mostly close to Bradyrhizo-bium sp. were present in all but one UPW systems. Previousresearch showed that various representatives of the genusPseudomonas are present in UPW (6, 16, 22, 33). P. aeruginosaand Burkholderia cepacia were shown to contaminate a water-distilling system (13). R. pickettii strains were also reportedpresent 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 typicalbacterial 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 thesebacteria on standard R2A media; ASTM guidelines (1, 2)

recommend growing the bacteria (for isolation from UPW) for48 to 72 h. Our experiments showed that this period of incu-bation is insufficient for growth of Bradyrhizobium sp. presentin UPW systems (18).

It is important to emphasize that very little variation inbacterial counts was observed when different parts of UPW

polishing loops were analyzed. This observation questions theeffectiveness of some stages of antibacterial treatment em-ployed in modern UPW production: in particular, UV254treatment seems to have little effect on the bacterial numberspresent in water. These findings may be better understood inconjunction with the nature of bacterial growth in UPW sys-tems, which according to most available evidence occurs in theform of biofilms attached to inner surfaces (11, 20). If we takeinto consideration the results of the present study, it may besuggested that each part of the UPW production system (sep-arated from others by filters, UV-units, etc.) possesses its ownbacterial 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 workmay be needed for its confirmation. It is worth noting thatBradyrhizobium strains were detected in all plants where nitro-gen had been used; however, the same group of bacteria wasalso isolated from the UPWS-2, which does not contain nitro-gen. The industrial plant seemingly free from Bradyrhizobiumsp. did not use nitrogen in its system (UPWS-4).

Discovery of nifH genes in Bradyrhizobium strains is notsurprising by itself, but when taken in conjunction with thespread of this bacterial group in UPW systems, it may suggestthat nitrogen contributes to cell maintenance and growth inthese systems. The presence ofnifHsequences in P. saccharo-philia is somewhat more unusual. However, a few examples ofnitrogen-fixing Pseudomonas strains have been reported (5, 15,36), and a nitrogen-fi xing strain of P. saccharophilia ATCC15946 has also been reported (3). It is important to stress thatfinding bacterial strains with nif genes deserves further inves-tigation, since it provides the first evidence that the widespreaduse of nitrogen in UPW production systems may contribute tobacterial contamination.

Identification of the typical bacterial strains inhabiting UPWsystems allowed us to design oligonucleotide primers specificto two main bacterial groups. PCR experiments conducted

with UPW samples demonstrated the possibility for detectionofR. 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 thesystem were too low to allow direct PCR detection of bacterial16S rDNA sequences. The use of specific (as well as universal)primers for the detection of the bacterial contamination inUPW systems may be considered useful for the preliminaryassessment of bacterial presence in UPW.

In conclusion, it should be said that certain bacterial popu-lations appear common to many industrial UPW systems andare 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 oligotrophicUPW environment.


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ACKNOWLEDGMENTS

We acknowledge the support of the NSF Industry/University Coop-erative Research Center Program and of the Industrial Research andTechnology Unit Northern Ireland START Programme Grant un-der the international TIE Project, Microbiocontamination in Ultra-pure Water, involving researchers at the University of Arizona, TheQueens University of Belfast, SUNY at Buffalo, and NJIT.

We also thank four industrial sites that allowed us to sample theirUPW systems and Jon Sjogren for help with the collection of UPWsamples.

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