Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems

Abstract

Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4,6-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (>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.

Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic

“…Diazotrophs are typically found in low abundance in marine systems, and a nested PCR protocol (455 amplification cycles) is required to amplify the nifH gene. Clone libraries generated using these amplicons may contain heterotrophic nifH sequences amplified from organisms or nucleic acids present in PCR reagents (Kulakov et al, 2002;Zehr et al, 2003a;Goto et al, 2005;Farnelid et al, 2009), high-purity water systems (Matsuda et al, 1996) or sampling equipment. It is insufficient to run negative controls, as amplification of contaminant nifH is sporadic and difficult to reproduce.…”

Biofilm Composition and Threshold Concentration for Growth of Legionella pneumophila on Surfaces Exposed to Flowing Warm Tap Water without Disinfectant

“…The colony counts of the biofilm were predominated by B. japonicum (Table S4), a slowly growing nitrogen-fixing bacterium and a root nodule microsymbiont of soybean (68). This amoeba-resistant bacterium and related species are frequently observed in tap water installations (66) and ultrapure water systems (69). Endosymbiotic growth within V. vermiformis (70) may explain its presence in the biofilm, but the symbiotic relationship is not yet understood.…”

Rapid Screening for Freshwater Bacterial Groups by Using Reverse Line Blot Hybridization

“…Still, the distribution in the size fractions is interesting, since in most positive samples, only the larger fraction was positive. One of the cultivated species targeted by the probe, Ralstonia pickettii, is occasionally found in clinical specimens (18,38) and is known to survive in aqueous solutions, both distilled water and saline (17,18). Other species from this cluster, such as Ralstonia solanacearum, are associated with plant disease (11,30).…”