E.coli and enterococci subtyping to discriminate contamination sources in wastewater treatment ponds

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Arnau Casanovas-Massana (Yale School of Public Health)Anicet Blanch (University of Barcelona)


The use of reclaimed water to irrigate golf courses has increased notably in the last years due to the availability of this resource during dry periods when other conventional resources are scarcer. To take advantage of this alternative resource, a common practice in golf courses is the construction of open-air ponds within its premises. These ponds serve not only as water reservoirs, but they have also landscaping and beautification purpose. The reclaimed water used to fill these ponds comes usually from urban wastewater treatment plants that include a final disinfection step. However, it has been suggested that the microbial faecal populations present in the reclaimed water after disinfection may regrow in the ponds, and/or the ponds may be recontaminated with new faecal material contributions. In this case-study, an investigation conducted in two reclaimed water open-air ponds used to irrigate a golf course in North-eastern Spain is presented. The aim of this study was to determine whether the low levels of faecal indicator bacteria (FIB) detected in the aforementioned ponds were due to a regrowth of the reclaimed water populations or to an input of faecal material whose source was in the golf facility. To this end, three hundred and fifty enterococcal strains and 308 faecal coliform strains were isolated from the ponds and reclamation plant in two sampling campaigns (summer and winter), and they were biochemically phenotyped. In addition, the inactivation of several microbial faecal pollution indicators (faecal coliforms, total bifidobacteria, sorbitol-fermenting bifidobacteria, somatic bacteriophages, and bacteriophages infecting Bacteroides thetaiotaomicron) was studied using a mesocosm in situ in order to obtain information about their potential regrowth under field conditions. The obtained results showed that although FIB concentration was low, the biochemical fingerprinting indicated that the origin of the faecal contamination in the ponds was not related to the reclaimed water, but to a source related to the pond’s own dynamics, most likely migrating birds. Furthermore, the mesocosm assays indicated that none of the microbial faecal indicators was able to regrow in the ponds in any of the seasons, although their persistence significantly increased in the winter. Overall, this case-study highlights the fact that reclaimed water may be recontaminated in open-air reservoirs, and therefore, its microbial quality should be monitored throughout its use.