Occurrence of Sphingomonas sp. bacteria in cold climate Water Science and Technology: Supply drinking water supply system biofilms P. Vuoriranta, M. Männistö and H. Soranummi Tampere University of Technology, Institute of Environmental Engineering and Biotechnology, P.O. Box 541, FIN-3310 Tampere, Finland (E-mail: pertti.vuoriranta@tut.fi) Abstract Members of the bacterial genus Sphingomonas (recently split into four genera), belonging to α-4-subclass of Proteobacteria, were isolated and characterised from water distribution network biofilms. Water temperature in the studied network, serving 200,000 people, is less than 5°C for about five months every winter. Sphingomonads, characterised using fluorescent oligonucleotide probes and fatty acid composition analysis (FAME), were a major group of bacteria among the distribution network biofilm isolates. Intact biofilms, grown on steel slides in a biofilm reactor fed with tap water, were detected in situ using fluorescence labelled oligonucleotide probes (FISH). Hybridisation with probes targeted on α- Vol 3 No 1–2 pp 227–232 proteobacteria and sphingomonads was detected, but FISH on intact biofilms suffered from non-specific hybridisation and intensive autofluorescence, possibly due to extracellular material around the bacterial cells attached to biofilm. These preliminary results indicate that bacteria present in the distribution network biofilms in this study phylogenetically differ from those detected in more temperate regions. Keywords Drinking water; FAME; FISH; proteobacteria; Sphingomonas Introduction Water supply systems, e.g. water treatment plants, distribution networks, water towers or © IWA Publishing 2003 respective constructions, and finally installations serving single households or enterprises, offer a variety of ecological niches for microbes and their predators (Kalmbach et al., 1997). The conventional way to survey water microbiology, i.e. hygienic water analysis, has a limited aim of revealing faecal contamination. Heterotrophic plate count, often included in water quality survey, gives quantitative information about easily culturable water bacteria. Most bacteria in oligotrophic habitats, such as distribution networks, grow very slowly, or do not grow at all on any standard growth medium. Microscopic techniques, such as direct count using dyes labelling every bacterial cell (DAPI, AO), may be used to obtain total cell counts. Total counts, however, may remain relatively constant even when population dynamic drifts, affecting water supply system condition or consumer health, take place. Furthermore, conventional control of drinking water quality does not consider bacteria attached to the biofilms growing on any surface in contact with water. Recently, methods based on characterisation and comparison of bacterial nucleic acids seem to offer an increasingly common outcome from the problem of surveying water and biofilm bacteria not only by quantity but quality as well (Manz et al., 1992, 1993; Schwartz et al., 1998; Amann et al., 2001). To get data for a rational design, operation and optimisation of water treatment pro- cesses and network management, a project aiming at a better understanding of bacterial diversity and population dynamics in water supply systems was launched. Emphasis was put on methods allowing rapid detection of a selected bacterial group, the sphingomonads, in water supply system biofilms. The nomenclature of the genus Sphingomonas has been recently revised and the genus divided into four genera: Sphingomonas, Sphingobium, Novosphingobium and 227 Sphingopyxis (Takeuchi et al., 2001). These genera belong to the α-4 subgroup of Proteobacteria and are in this paper referred to as sphingomonads. Sphingomonads were selected as some members of this group are known to be pathogens or opportunistic pathogens, they produce extracellular polymers, enhancing biofilm formation and resistance towards oxidants used for disinfection, and some of them are known to induce corrosion in copper pipes (Busse et al., 1999; White et al., 1996). P. Vuoriranta Material and methods Water quality and biofilm sampling Biofilms from the distribution network were collected from water meters at household con- et al. nections. Biofilm detachment was enhanced by ultrasonication (48 kHz, 4 × 1 min) in a Tris buffer containing zwittergent (Camper et al., 1985). Biofilms for in situ detection were grown in a biofilm reactor fed with tap water. Hydraulic retention time in the reactor was 12 hours. Substrata were steel slides (AISI 316). Tap water in the study area is almost exclusively originating from a boreal lake, rich in humic substances. Water treatment process consists of coagulation–flocculation, floata- tion, activated carbon filtration and disinfection with chlorine and chlorine dioxide. Isolation of α-4 proteobacteria from drinking water Serial dilutions of the bacterial suspensions, detached from a water meter, were plated on R2A agar (Reasoner and Geldreich, 1985) and incubated at 20°C for 21 d. Randomly chosen colonies were picked from the plates and streaked on to new R2A plates. A total of 51 strains were isolated. In situ hybridisation of the isolated strains To investigate the occurrence of Sphingomonas species among the isolated strains, the iso- lates were hybridised with CY3 labelled probes ALF4-1322 and SPH120 specific for α-4 subgroup of Proteobacteria and Sphingomonas species, respectively (Neef, 1997). For hybridisation, the isolates were grown on R2A broth overnight. Cells were centrifuged, washed once with phosphate buffered saline, fixed for 3 h at 4°C and hybridised with CY3- labelled probes for 1.5 h at 46°C as described by Manz et al. (1992). The probe concentra- tion was 5 ng/µl and formamide concentration as shown in Table 2. In situ hybridisation of biofilms For the fixation and hybridisation of biofilm samples, the steel slides were partitioned into three sections with a waterproof pen and a string of adhesive tape. The sections were fixed at 4°C by adding 50 µl of p-formaldehyde (4%). After 1.5 h fixation the fixative was care- fully removed and the slides rinsed with distilled water and air dried. Hybridisation was Table 1 Quality of treated lake water at the waterworks n mean min. max. Temperature (°C) 251 8.4 1.2 19.5 pH 248 8.5 7.8 9.0 DOC (mg/)L 98 2.9 2.4 3.4 µ < < Ptotal ( g/L) 12 3 2 8.0 µ < < NH4-N ( g/L) 12 5 1 8.0 A254 23 0.029 0.022 0.038 Alkalinity (mmol/L) 249 0.73 0.57 0.99 Hardness (mmol/L) 51 3.3 3.0 3.6 Total chlorine (mg/L) 251 0.40 0.35 0.51 HPC(TGY) (22°C 3d) 13 0 0 1 228 Table 2 Oligonucleotide probes and hybridisation conditions applied in this study Probe Specificity Sequence (5’ ( 3’) FA %a Reference EUB338 Bacteria GCTGCCTCCCGTAGGAGT 20 Schramm et al., 1996 Alf 1b α-Proteobacteria CGTTCGYTCTGAGCCAG 20 Manz et al., 1992 Alf4-1322 α-4 Proteobacteria TCCGCCTTCATGCTCTCG 40 Neef, 1997 SPH120 Sphingomonas GGGCAGATTCCCACGCGT 35 Neef, 1997 BET42a β-Proteobacteria GCCTTCCCACTTCGTTT 35 Manz et al., 1992 P. Vuoriranta CF319a Cytophaga-Flavobacterium TGGTCCGTGTCTTCAGTAC 35 Manz et al., 1996 GAM42a γ-Proteobacteria GCCTTCCCACATCGTTT 35 Manz et al., 1992 NON338 ACTCCTACGGGAGGCAGC 20 Manz et al., 1992 a Percentage of formamide in in situ hybridisation buffer et al. performed as described for the isolates by adding 50 µl of hybridisation buffer and 5 ng/µl of CY3-labelled probes to the sections. Whole cell fatty acid composition of the isolated strains To confirm the positive hybridisation results, whole cell fatty acids of isolates that hybridised with ALF4-1322 and SPH120 probes were analysed. For the analysis, cells were grown on R2A agar at 20°C for 3 days. Fatty acids were methylated, extracted and analysed by GC-FID as described by Männistö and Puhakka (2001). The presence of high amounts (> 30%) of 18:1 and 14:0-2OH fatty acids was considered an indicator that the isolate is a member of the genus Sphingomonas. Results and discussion Biofilm isolates Colonies formed on R2A from dilutions of detached water meter biofilm suspensions were overwhelmingly smooth and colourful, colours ranging from yellow to orange (Figure 1). Colonies for isolates were picked at random. Out of 51 isolates, 35 hybridised with oligonucleotide probe Alf4-1322, targeting on α-4-proteobacteria, and 32 hybridised with SPH120, targeting on Sphingomonas, the genus later on having been split into several new genera. Colonies of hybridised isolates were pigmented, many of them in various tones of orange. Twenty-nine of the random isolates hybridised with both Alf4-1322 and SPH120. These isolates were exposed to whole cell fatty acid analysis (FAME). Twenty-six out of the 29 isolates contained 14:0 2OH and high amounts of 18:1 fatty acids, characteristic to sphingomonads. Three isolates that hybridised with both probes were rejected from further studies, once iso- and anteiso fatty acids, referring to Gram-positive bacteria, were found. A dendrogram based on FAME compositions is shown in Figure 2. a b c Figure 1 Spread plate on R2A (a) and 1/5R2A (b) of detached biofilm from a water meter. A yellow isolate on R2A (c) 229 26 3 27 17 37 14 40 34 39 31 P. Vuoriranta 18 36 10 8 13 41 et al. 5 4 46 16 9 23 25 2 21 30 0 1 2 3 4 5 6 78 Euclidian distance Figure 2 Sphingomonad isolates from a water meter biofilm, related with respect to whole cell fatty acid compositions In situ biofilm hybridisation Steel slides, incubated in the biofilm reactor for 3 weeks were hybridised with CY-labelled EUB338, Alf1b, Alf4-1322, SPH120, BET42a and GAM42a probes. Hybridisation with EUB338 yielded generally high numbers of fluorescent cells or particles. When compared to the DAPI counts, the amount of EUB338 signals were typically close to 100% or even higher of the total number of bacteria. Generally the percentage of hybridised cells is sig- nificantly less than that of DAPI counts (Glöckner et al., 1999) which indicates that some of the signals detected here were due to unspecific hybridisation or autofluorescence.