# eDNA- and eRNA-Based Detection of 2-Methylisoborneol-Producing Cyanobacteria and Intracellular Synthesis Dynamics in Freshwater Ecosystem

## TL;DR

This study shows that monitoring gene activity in water can predict unpleasant odors from cyanobacteria weeks before they occur, helping to manage drinking water quality.

## Contribution

The study introduces eRNA monitoring as an early warning system for 2-MIB-related odors in freshwater.

## Key findings

- eRNA levels of the mibC gene preceded 2-MIB concentration increases by 2–4 weeks.
- eRNA monitoring showed a strong correlation with 2-MIB concentrations (r = 0.879).
- Combining eDNA and eRNA analyses improves the accuracy of predicting odor events.

## Abstract

Off-flavors and odors in drinking water sources are often linked to the occurrence of cyanobacteria. Specific species produce the secondary metabolite 2-methylisoborneol (2-MIB), which causes a distinct earthy–musty odor that can be detected even at concentrations below the nanogram-per-liter level. Predicting the occurrence of 2-MIB remains difficult due to its sporadic production, which is influenced by both physiological state and environmental factors. Conventional monitoring approaches typically identify the compound only once accumulation is underway. In this study, we performed a longitudinal survey along the North Han River, Republic of Korea, to assess both the presence of 2-MIB-producing cyanobacteria and their active 2-MIB biosynthesis. Biweekly water samples were collected from twelve locations for over two years, and analyses included both 2-MIB concentrations and the expression levels of the biosynthetic genes. Notably, heightened expression of 2-MIB biosynthetic genes was observed approximately two to four weeks before increases in detected 2-MIB concentrations. Our results indicate that monitoring biosynthetic gene expression at the molecular level provides an effective early warning for odor episodes in drinking water supplies. Applying this approach would allow water resource managers to initiate timely interventions, ultimately protecting water quality and reducing complaints from consumers.

Taste and odor (T&O) compounds in freshwater are frequently produced by certain cyanobacteria; however, their occurrence remains difficult to predict. This study examined the temporal and spatial variations in the mibC gene, which encodes a critical enzyme in the biosynthesis of 2-methylisoborneol (2-MIB), by analyzing environmental DNA (eDNA) and RNA (eRNA) in the North Han River, Republic of Korea, from July 2019 to October 2021. Surface water was sampled at twelve sites and analyzed for mibC DNA copy number, RNA expression, cyanobacterial cell density, and 2-MIB concentration using quantitative PCR (qPCR), microscopy, and gas chromatography–mass spectrometry (GC–MS). The mibC gene was present throughout the year, exhibiting peaks from late summer to early winter; higher concentrations typically initiated upstream and subsequently moved downstream. RNA expression was elevated from summer to autumn, rapidly declined following heavy rainfall, and reliably preceded increases in 2-MIB concentrations by 2–4 weeks. RNA levels were strongly correlated with 2-MIB concentrations (r = 0.879, p < 0.001) but showed only a moderate association with Pseudanabaena cell density, whereas DNA demonstrated weaker correlations. More than 95% of total 2-MIB was dissolved, limiting the ability to directly estimate concentrations from eRNA data alone. The results indicate that eRNA monitoring is an effective early warning tool for T&O events. In addition, combining eDNA and eRNA analyses enables a more accurate evaluation of T&O-producing cyanobacteria, presenting practical benefits for proactive management of drinking water.