Isolation and Characterization of Cylindrospermopsis raciborskii Strains from Finished Drinking Water

Abstract

In the summer of 2015, an intense cyanobacterial bloom producing geosmin/2-methylisoborneol (MIB) occurred in the Roxo freshwater reservoir in Alentejo, Portugal. The drinking water supplied from the Roxo water treatment plant (WTP) exhibited an unpleasant odor/taste and a significant cyanobacteria density was detected in the finished water at the exit of the WTP. Cyanobacteria were not evaluated downstream of the WTP, namely, at the city reservoir. The aim of this work was to isolate and characterize viable cyanobacteria present in finished water (exit of the WTP and city reservoir) that withstand conventional water treatment. Treated water samples collected at both sites were inoculated in Z8 culture medium to provide the conditions for putative cyanobacterial growth. After 30 days, filamentous cyanobacteria were observed in cultures inoculated with samples from the exit point of the WTP. Viable trichomes were isolated and identified as Cylindrospermopsis raciborskii by morphometric and molecular analysis. None of the isolates were cylindrospermopsin/microcystin producers, as confirmed by ELISA and amplification of corresponding genes (PS/PKS and mcyA-cd/mcyAB/mcyB). ELISA results were positive for saxitoxin, but saxitoxin and derivatives were not detected by liquid chromatography with fluorescence detection (LC-FLD), nor were their related genes (sxtA/sxtA4/sxtB/sxtM/sxtPer/sxtI). To our knowledge, this is the first report on the establishment of cultures of C. raciborskii that resisted water treatment processes.

Keywords: Cylindrospermopsis raciborskii; culture collection; finished water; saxitoxin.

Figure 1

Morphological characteristics of C. raciborskii isolates LMECYA 324 (A), LMECYA 325 (B), and LMECYA 326 (C). Microphotographs from live (A1, B1–3 and C1–2) and Lugol’s-fixed cultures (A2–7, B4–7 and C3–7). Magnification 400× (A1–4, B1, B4–5, and C1, C3–5) and 1000× (A5–7, B2–3, B6–7 and C2, C6–7). Scale bar = 10 µm.

Figure 2

Distribution of the length (µm) (a) and width (µm) (b) of vegetative cells, heterocytes, and akinetes from C. raciborskii LMECYA 324 (green), LMECYA 325 (orange), and LMECYA 326 (violet). The box boundaries indicate the 25th and 75th percentiles. The line within the box marks the median and the black dots indicate outliers.

Figure 3

Phylogenetic tree of C. raciborskii LMECYA 324–326 and related taxa obtained by the maximum likelihood method of the 16S rDNA. An alignment of 540 positions in the final dataset was used. Percentage bootstrap values of 1000 replicates are given at each node. GenBank accession numbers are indicated after species designation (names in bold-face correspond to the sequence determined in this study).