Investigation of Natural Biofilms Formed During the Production of Drinking
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ARTICLE IN PRESS Water Research 38 (2004) 1197–1206 Investigation of natural biofilms formed during the production of drinking water from surface water embankment filtration Farahnaz Emtiazi, Thomas Schwartz*, Silke Mareike Marten, Peter Krolla-Sidenstein, Ursula Obst Department of Environmental Microbiology, Forschungszentrum Karlsruhe GmbH, Institute for Technical Chemistry–Water Technology and Geotechnology Division, P.O. Box 3640, Karlsruhe D-76021, Germany Received 5 June 2003; received in revised form 8 October 2003; accepted 13 October 2003 Abstract Populations of bacteria in biofilms from different sites of a drinking water production system were analysed. Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analyses revealed changing DNA band patterns, suggesting a population shift during bank filtration and processing at the waterworks. In addition, common DNA bands that were attributed to ubiquitous bacteria were found. Biofilms even developed directly after UV disinfection (1–2 m distance). Their DNA band patterns only partly agreed with those of the biofilms from the downstream distribution system. Opportunistic pathogenic bacteria in biofilms were analysed using PCR and Southern blot hybridisation (SBH). Surface water appeared to have a direct influence on the composition of biofilms in the drinking water distribution system. In spite of preceding filtration and UV disinfection, opportunistic pathogens such as atypical mycobacteria and Legionella spp. were found in biofilms of drinking water, and Pseudomonas aeruginosa was detected sporadically. Enterococci were not found in any biofilm. Bacterial cell counts in the biofilms from surface water to drinking water dropped significantly, and esterase and alanine-aminopeptidase activity decreased. b-glucosidase activity was not found in the biofilms. Contrary to the results for planktonic bacteria, inhibitory effects were not observed in biofilms. This suggested an increased tolerance of biofilm bacteria against toxic compounds. r 2003 Elsevier Ltd. All rights reserved. Keywords: Biofilms; Population shifts; Opportunistic pathogenic bacteria; Enzyme activities; Molecular-biological techniques 1. Introduction reduced and eliminated. Research results obtained with respect to the hydraulic, physico-chemical, and chemi- Bank filtration and artificial groundwater enrichment cal/biological processes revealed a good and stable long- are frequently employed for the production of drinking term cleaning efficiency of bank filtration. This also water. When surface water enters the aquifer via an applies to the removal of particles, pathogens, a number underground passage due to potential gradients gener- of organic, and most trace substances [1] except for some ated by wells, this is referred to as bank filtration. individual organic polar compounds which are persis- During underground passage, a variety of chemical and tent in a nearly unrestricted manner [2]. Microorganisms biological processes occur, by which compounds are significantly contribute to cleaning during the under- ground passage by enzymatic degradation or partial *Corresponding author. Tel.: +49-7247-826-802; fax: +49- metabolism of water impurities and by physico-chemical 7247-826-858. processes, e.g. adsorption. However, specific manipula- E-mail address: [email protected] tion and use of these microbial elimination processes (T. Schwartz). have failed, and knowledge of the processes is too 0043-1354/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2003.10.056 ARTICLE IN PRESS 1198 F. Emtiazi et al. / Water Research 38 (2004) 1197–1206 incomplete. Still these biological processes during 2. Material and methods natural underground passage are suited as environmen- tally compatible and low-cost stage for sustainable 2.1. Biofilm sampling method drinking water processing. This is not only true for Central Europe with its large river basins, but also for For biofilm formation in drinking water distribution many Third World and threshold countries that have to systems, special devices were used [6,7]. The core of these produce drinking water from highly contaminated devices consisted of a hollow stainless steel cylindrical surface waters. Yet few data are available for the element, where stainless steel bolts holding steel platelets comparative description of function and population for biofilm growth were screwed into place. The platelet changes of adhesive bacteria (biofilms) during the (15 mm  30 mm  2.5 mm) was attached to the end of a underground passage and in the downstream drinking bolt with a small screw. To study natural biofilms, these water production facilities. devices were installed at different sampling points in the Among the major ecological units of aquatic systems, waterworks, i.e. downstream of the granular activated which affect water quality, are biofilms that cover carbon filtration (GAC) and downstream of UV practically all accessible wet surfaces. Biofilms may be disinfection (DIS). Two devices were installed in house composed of algae, bacteria, fungi and other eukaryotic branch connections within the municipal drinking water microorganisms, and cover surfaces, e.g. in storage distribution system (DW1 and DW2) 1–2 km down- basins, filter systems, and drinking water distribution stream of the waterworks. Additionally, platelets lines. As biofilms represent the predominating biological (170 mm  20 mm  2 mm) were incubated in surface form of life in habitats of water and soil, it is urgently water used for embankment filtration (SW, Rhine river) required to improve the understanding of the structure and raw water (RW) from a well downstream of bank and function of these biocoenoses. There is a lack of filtration. To examine the biofilm populations, the methods for the detection of adhesive bacteria in platelets were removed after 3–4 weeks, the biofilms classical drinking water analysis. Cultivation processes were scraped from the surface using a sterile cell scraper cover a small part of the natural planktonic population (Nunc, Wiesbaden, Germany) and suspended in 10 ml only [3]. The limitation of nutrients and environmental water from the sampling site. The biofilm suspensions stress situations may induce physiological and morpho- were centrifuged for 10 min at 10 000g. The pellet was logical changes in many aquatic bacteria. A dormant resuspended in 1 ml sterile water. DNA was extracted status was described for pathogenic bacteria, such from biofilms from the surface water and horizontal well as Campylobacter spp., E. coli, and Legionella pneumo- (QIAamps DNA Mini Kit-50, Qiagen). These DNA phila [4,5] which makes their isolation and identi- preparations were used as template in PCR. The biofilm fication problematic. Therefore, cultivation-independent suspensions from GAC, DIS, and DW were used molecular-biological methods targeting nucleic acids are without extraction for subsequent molecular-biological required in addition to biochemico-metabolic analyses analysis. [5]. Our investigations covered biofilms from surface 2.2. PCR-DGGE analysis of different aquatic biofilms water, raw water after bank filtration, processed drinking water prior to and after UV disinfection as PCR primers targeting the regions (V1-3) of 16S well as from the downstream municipal distribution rDNA of bacteria were used for biofilm analysis system. As an alternative to classical cultivation (Table 1) [8,9]. For DGGE analysis, a sequence of methods, molecular-biological methods were applied multiple guanines (G) and cytosines (C) was attached to with the DNA as target molecule, which exists in each the 50end of the forward primer [10]. A ‘‘touch-down’’ cell irrespective of its physiological state. Oligonucleo- PCR profile published by Kilb et al. [9] was applied. The tide probes developed for a number of environmental final 100 ml reaction mixture contained 2.5 U of HotStar bacteria and pathogens allowed to comprehensively Taq-DNA polymerase (Qiagen, Germany), 30 pmol of describe bacterial populations or identify pathogens each primer, 1  PCR buffer, 1.5 mM MgCl2, 200 mM without a cultivation pre-enrichment. By means of the dNTPs, and 10 ml biofilm suspension or template DNA. polymerase chain reaction (PCR), denaturing gradient A GeneAmp PCR System 9700 (Applied Biosystems) gel electrophoresis (DGGE), and southern blot hybridi- was used for PCR. Aliquots of 5 ml were analysed by sation (SBH), population shifts of the bacterial biofilms electrophoresis in a 1% agarose gel containing ethidium were investigated and the occurrence of facultatively bromide to check the sizes and amounts of the pathogenic bacteria, such as legionellae, mycobacteria, amplicons. PCR products were purified using phenol- and enterococci, was studied. Enzyme activities were chloroform-isoamyl alcohol (25:24:1 vol.), precipitated measured in biofilm samples to determine the metabolic with isopropanol, washed with 70% ethanol, dried, and performance and physiological states of the bacterial resuspended in 20 ml sterile water. DGGE analysis of biocoenoses. PCR products (B526 bp and 349 bp) was performed ARTICLE IN PRESS F. Emtiazi et al. / Water Research 38 (2004) 1197–1206 1199 Table 1 PCR primers and SBH probes used Primer/probes Organism Sequence (50-30) Target site Reference Primer GC27Fa Bacteria AGAGTTTGATCMTGGCTCAGb 8–27 (16S rDNA) [9] Primer 517R Bacteria ATTACCGCGGCTGCTGG 534–517 (16S rDNA) [9] Primer 342R Bacteria CTGCTGCCTCCCGTAG 357–342 (16S rDNA) [8] Primer-Eub338F Bacteria ACTCCTACGGGAGGCAGC