Drinking water biofilms on copper and stainless steel exhibit specific molecular responses towards different disinfection regimes at waterworks.

Biofilms growing on copper and stainless steel substrata in natural drinking water were investigated. A modular pilot-scale distribution facility was installed at four waterworks using different raw waters and disinfection regimes. Three-month-old biofilms were analysed using molecular biology and microscopy methods. High total cell numbers, low counts of actively respiring cells and low numbers of cultivable bacteria indicated the high abundance of viable but not cultivable bacteria in the biofilms. The expression of the recA SOS responsive gene was detected and underlined the presence of transcriptionally active bacteria within the biofilms. This effect was most evident after UV disinfection, UV oxidation and UV disinfection with increased turbidity at waterworks compared to chemically treated and non-disinfected systems. Furthermore, live/dead staining techniques and environmental scanning electron microscopy imaging revealed the presence of living and intact bacteria in biofilms on copper substrata. Cluster analyses of DGGE profiles demonstrated differences in the composition of biofilms on copper and steel materials.

UV-induced dark repair mechanisms in bacteria associated with drinking water.

Caulobacter crescentus and Aquabacterium commune, both isolated from drinking water, as well as environmental isolates of Pseudomonas aeruginosa and Enterococcus faecium were treated with different UV fluences to study their capacity to restore induced DNA damages. Here, the induction of a key mechanism of bacterial dark repair, the so-called recA system, was analysed. With newly designed probes, the specific recA mRNA was detected by Northern blot. Additionally, the RecA protein was measured by the Western blot technique using a specific antibody. In drinking water bacteria as well as in opportunistic microorganisms, a specific induction of dark repair mechanisms was found even at UV fluences higher than 400J/m(2), the German standard for UV disinfection. This induction depended on the incubation time after UV treatment. Nevertheless, the UV-induced recA expressions were found to differ in the bacteria under investigation.

DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses.

Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems.

Combined use of molecular biology taxonomy, Raman spectrometry, and ESEM imaging to study natural biofilms grown on filter materials at waterworks.

DNA-based population analysis was applied in combination with Raman spectrometry and Environmental Scanning Electron Microscopy for the characterisation of natural biofilms from sand and activated carbon filters operated for a long term at a municipal waterworks. Whereas the molecular biology polymerase chain reaction combined with denaturing gradient gel electrophoresis approach provides a deeper insight into the bacterial biofilm diversities, Raman spectrometry analyses the chemical composition of the extracellular polymer substances (EPS), microorganisms embedded in EPS as well as other substances inside biofilm (inorganic compounds and humic substances). Microscopy images the spatial distribution of biofilms on the two different filter materials. In addition, bacterial bulk water populations were compared with biofilm consortia using the molecular fingerprint technique mentioned. Population analysis demonstrated the presence of more diverse bacterial species embedded in a matrix of EPS (polysaccharides, peptides, and nucleic acids) on the sand filter materials. In contrast to this, activated carbon granules were colonised by reduced numbers of bacterial species in biofilms. Besides alpha-, beta-, and gamma-Proteobacteria, a noticeable specific colonisation with Actinobacteria was found on activated carbon particles. Here, the reduced biofilm formation came along with a decreased EPS synthesis. The taxonomy profiles of the different biofilms revealed up to 60% similarity on the same filter materials and 32% similarity of different materials. Similarity of adherent communities from filter materials and bulk water populations from the filter effluent varied between 36% and 58% in sand filters and 6-40% in granular activated carbon filters. The biofilm investigation protocols are most crucial to subsequent acquisition of knowledge on biofilm dynamics and bacterial contributions to transformation or adsorption processes in waterworks facilities.