First evidence of off-flavour haloanisoles production by dormant fungal spores in drinking water: Formation pattern, influencing factors and transcriptome analysis.

Fungi are major producers of off-flavour haloanisoles (HAs) in drinking water supply systems. However, evidence of HA production by dormant fungal spores remains unclear. This study investigated the microbial O-methylation of dormant spores of a fungus Aspergillus sp. from drinking water treatment plants. Dormant spores were capable of O-methylating 2,4,6-trichlorophenol (2,4,6-TCP) to produce 2,4,6-trichloroanisole (2,4,6-TCA). A pronounced linear correlation was identified between the precursor load and the 2,4,6-TCA formation capability, with a function lg(y)= 0.542lg(x) - 4.28 (R = 0.838). The effects of metal ions on fungal spore O-methylation were multifaceted and highly concentration-dependent. Natural organic matters could inhibit 2,4,6-TCA formation. NHCl presented higher efficacy than free chlorine in inhibiting spore O-methylation. However, O-methylation inhibition rates were lower than spore inactivation rates, suggesting spores in viable but non-culturable state still possessed partial ability to form 2,4,6-TCA. Transcriptome analysis revealed that 50 nM 2,4,6-TCP treatment had no impact on the spore's transcriptional profile, whereas 1 mM Cu treatment significantly inhibited O-methylation process by down-regulating genes involved in ATP generation. This study provides a first evidence that dormant fungal spores are capable of producing 2,4,6-TCA, raising concerns about the potential off-flavour risk and the development of targeted control strategies in drinking water systems.