Evaluation of DNA extraction yield from a chlorinated drinking water distribution system

Desalination technology based on Reverse Osmosis (RO) membrane filtration has been resorted to provide high-quality drinking water. RO produced drinking water is characterized by a low bacterial cell concentration. Monitoring microbial quality and ensuring membrane-treated water safety has taken advantage of the rapid development of DNA-based techniques. However, the DNA extraction process from RO-based drinking water samples needs to be evaluated regarding the biomass amount (filtration volume) and residual disinfectant such as chlorine, as it can affect the DNA yield. We assessed the DNA recovery applied in drinking water microbiome studies as a function of (i) different filtration volumes, (ii) presence and absence of residual chlorine, and (iii) the addition of a known Escherichia coli concentration into the (sterile and non-sterile, chlorinated and dechlorinated) tap water prior filtration, and directly onto the (0.2 μm pore size, 47 mm diameter) mixed ester cellulose membrane filters without and after tap water filtration. Our findings demonstrated that the co-occurrence of residual chlorine and low biomass/cell density water samples (RO-treated water with a total cell concentration ranging between 2.47 × 102–1.5 × 103 cells/mL) failed to provide sufficient DNA quantity (below the threshold concentration required for sequencing-based procedures) irrespective of filtration volumes used (4, 20, 40, 60 L) and even after performing dechlorination. After exposure to tap water containing residual chlorine (0.2 mg/L), we observed a significant reduction of E. coli cell concentration and the degradation of its DNA (DNA yield was below detection limit) at a lower disinfectant level compared to what was previously reported, indicating that free-living bacteria and their DNA present in the drinking water are subject to the same conditions. The membrane spiking experiment confirmed no significant impact from any potential inhibitors (e.g. organic/inorganic components) present in the drinking water matrix on DNA extraction yield. We found that very low DNA content is likely to be the norm in chlorinated drinking water that gives hindsight to its limitation in providing robust results for any downstream molecular analyses for microbiome surveys. We advise that measurement of DNA yield is a necessary first step in chlorinated drinking water distribution systems (DWDSs) before conducting any downstream omics analyses such as amplicon sequencing to avoid inaccurate interpretations of results based on very low DNA content. This study expands a substantial source of bias in using DNA-based methods for low biomass samples typical in chlorinated DWDSs. Suggestions are provided for DNA-based research in drinking water with residual disinfectant.

Aeromonas species from non-chlorinated distribution systems and their competitive planktonic growth in drinking water

Aeromonas is included in the Dutch Drinking Water Decree as an indicator for elevated microbial regrowth in non-chlorinated drinking water distribution systems (DWDS). The temporal and spatial diversity of Aeromonas species in ten DWDS and their planktonic growth characteristics for different carbon sources was investigated. Genotyping of the gyrB gene of isolates showed a non-systematic temporal and spatial variable prevalence of seven different Aeromonas species in these DWDS and no correlation with AOC-P17/NOX and Aeromonas concentrations. Pure cultures of these seven species showed a high affinity to low concentrations (μg/L) of individual amino acids and fatty acids, compounds associated with biomass. Growth occurred at 0.5 μg-C/L of an amino acid mixture. Growth of a mixed community of A. rivuli, A. salmonicida, A. sobria and A. veronii in drinking water occurred in pasteurized samples, however, no growth and decay occurred in competition with the autochthonous bacteria (non-pasteurized samples). This community also failed to grow in non-pasteurized distribution samples from a location with clear increase in planktonic Aeromonas concentrations in the transported drinking water. For competitive planktonic growth of Aeromonas an amino acid concentration of ≥5 μg-C/L is required. AOC-P17/NOX concentrations showed that such concentrations are not expected in Dutch drinking water. Therefore, we suspect that competitive planktonic growth is not the major cause of the observed non-compliance with the Aeromonas standard in non-chlorinated DWSD. Importance The occurrence of the bacterial genus Aeromonas in non-chlorinated drinking water in the Netherlands is regarded as an indication for elevated microbial regrowth in the distribution system. Identification of the prevalent species in ten distribution systems by genotyping yielded seven different species, with A. rivuli, A. veronii and A. sobria as the most dominant ones. Planktonic growth experiments of pure cultures confirmed former published affinity of Aeromonas for certain biomass compounds (amino and fatty acids). In competition with the autochthonous microflora, however, planktonic growth was not observed, only after addition of a threshold amino acid concentration of 5 μg-C/L. Based on our results and further observations we deduced that planktonic growth of Aeromonas in the DWDS is not very likely. Benthic growth in loose deposits and planktonic release is a more plausible explanation for the observed planktonic increase of Aeromonas.