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Abstract2,4-Dichlorophenoxyacetic acid (2,4-D) is the third most applied pesticide in Brazil to control broadleaf weeds in crop cultivation and pastures. Due to 2,4-D’s high mobility and long half-life under anoxic conditions, this herbicide has high probability for groundwater contamination. Bioremediation is an attractive solution for 2,4-D contaminated anoxic environments, but there is limited understanding of anaerobic 2,4-D biodegradation. In this study, methanogenic enrichment cultures were obtained from Amazonian top soil (0—40 cm) and deep soil (50 -80 cm below ground) that biotransform 2,4-D (5 µM) to 4-chlorophenol and phenol. When these cultures were transferred (10% v/v) to fresh medium containing 40 µM or 160 µM 2,4-D, the rate of 2,4-D degradation decreased, and biotransformation did not proceed beyond 4-chlorophenol and 2,4-dichlorophenol in the top and deep soil cultures, respectively. 16S rRNA gene sequencing and qPCR of a selection of microbes revealed no significant enrichment of known organohalide-respiring bacteria. Furthermore, a member of the genus Cryptanaerobacter was identified as possibly responsible for phenol conversion to benzoate in the top soil inoculated culture. Overall, these results demonstrate the effect of 2,4-D concentration on biodegradation and microbial community composition, which are both important factors when developing pesticide bioremediation technologies.

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14Groundwater is a key water resource, with 45.7% of all drinking water globally being extracted from 15 groundwater. Maintaining good groundwater quality is thus crucial to secure drinking water. 16Micropollutants, such as pesticides, threaten groundwater quality which can be mitigated by 17 biodegradation. Hence, exploring microbial communities in aquifers used for drinking water 18 production is essential for understanding micropollutants biodegradation capacity. This study aimed 19 at understanding the interaction between groundwater geochemistry, pesticide presence, and 20 microbial communities in aquifers used for drinking water production. Two groundwater monitoring 21 wells located in the northeast of The Netherlands and at 500 m distance from each other were sampled 22Importance section 37 Groundwater is an essential source of drinking water. However, its quality is threathened by the 38 presence of micropollutants. Certain microorganisms are capable of degrading micropollutants. 39 However, groundwater is an unexplored environment, where the biodegradation potential of 40 naturally-present microorganisms is unknown. We thus explore how groundwater microbial ecology 41 in shaped by groundwater composition, namely geochemical parameters and micropollutants. This is 42 a first step towards understanding which microbial communities and environmental conditions 43 support natural attenuation of micropollutants. This study thus provides a first step towards 44 developing in situ bioremediation strategies to remove micropollutants from groundwater used for 45 drinking water production. 46 2.5 DNA extraction and quantification, PCR amplification and sequencing of the 16s rRNA gene, 131 and quantitative PCR (qPCR) 132 Microbial DNA was extracted from each filter using the DNeasy PowerSoil Kit (Qiagen, Hilden, 133 Germany) according to the manufacturer's instructions. The quality of DNA (average molecular size) 134

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