A microcystin synthesis mcyE/ndaF gene assay enables early detection of microcystin production in a tropical wastewater pond.

Cyanobacteria can dominate the algal community in wastewater ponds, which can lead to the production of cyanotoxins and their release into the environment. We applied traditional and molecular techniques to identify cyanotoxin hazards and high-risk periods in a tropical wastewater treatment system. Potentially toxic cyanobacteria were identified by microscopy and amplicon sequencing over the course of a year. Toxin gene levels were monitored and compared to toxin production to identify likely toxin producing species and high-risk periods. Cyanobacteria were persistent in the effluent year-round, with Planktothrix and Microcystis the most abundant genera; Microcystis could not be resolved beyond genus using amplicon sequencing, but M. flos-aquae was identified as a dominant species by microscopy. Microcystin toxin was detected for the first time in treated effluent at the beginning of the wet season (December 2018), which correlated with an increase in Microcystis amplicon sequence abundance and elevated microcystin toxin gene (mcyE/ndaF) levels. Concomitantly, microscopy data showed an increase in M. flos-aquae but not M. aeruginosa. These data informed a refined sampling campaign in 2019 and results showed a strong correlation between mcyE/ndaF gene abundance, microcystin toxin levels and Microcystis amplicon sequence abundance. Microscopy data showed that in addition to M. flos-aquae, M. aeruginosa was also abundant in February and March 2019, with highest levels coinciding with toxin detection and toxin gene levels. M. aeruginosa was the most abundant Microcystis species detected in selected treated effluent samples by metagenomics analysis, and elevated levels coincided with toxin production. All microcystin genes in the biosynthesis pathway were detected, but microcystin genes from Planktothrix agardhii were not detected. Gene toxin assays were successfully used to predict microcystin production in this wastewater system. Changes in amplicon sequence relative abundance were a useful indicator of changes in the cyanobacterial community. We found that metagenomics was useful not just for identifying the most abundant Microcystis species, but the detection of microcystin biosynthesis genes helped confirm this genus as the most likely toxin producer in this system. We recommend toxin gene testing for the early detection of potential toxin producing cyanobacteria to manage the risk of toxicity and allow the implementation of risk management strategies.