ORIGINAL PAPER 2,4-Dichlorophenoxyacetic acid degradation in methanogenic mixed cultures obtained from Brazilian Amazonian soil samples Gunther Brucha . Andrea Aldas-Vargas . Zacchariah Ross . Peng Peng . Siavash Atashgahi . Hauke Smidt . Alette Langenhoff . Nora B. Sutton Received: 3 June 2020 / Accepted: 31 March 2021 / Published online: 20 April 2021 Ó The Author(s) 2021 Abstract 2,4-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, metha- nogenic 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 lM) to 4-chlorophenol and phenol. When these cultures were transferred (10% v/v) to fresh medium contain- ing 40 lM or 160 lM 2,4-D, the rate of 2,4-D degradation decreased, and biotransformation did not proceed beyond 4-chlorophenol and 2,4-dichlorophe- nol 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 mem- ber of the genus Cryptanaerobacter was identified as possibly responsible for phenol conversion to ben- zoate in the top soil inoculated culture. Overall, these results demonstrate the effect of 2,4-D concentration on biodegradation and microbial community compo- sition, which are both important factors when devel- oping pesticide bioremediation technologies. Keywords Pesticide Á 2,4-Dichlorophenoxyacetic acid Á Methanogenic Á Biodegradation Á Soil Á Microbial diversity Introduction The Brazilian agriculture sector has undergone enor- mous growth in recent decades, with the country now becoming one of the world’s largest supplier of soy, coffee, sugar cane and corn, among other commodities (FAO 2015). However, this rapid growth has relied heavily on the use of pesticides. In 2008, Brazil became the world’s largest market for pesticide import, and in 2015, 899 million litres of pesticides Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10532-021-09940-3. G. Brucha Á A. Aldas-Vargas Á Z. Ross Á A. Langenhoff Á N. B. Sutton (&) Environmental Technology, Wageningen University & Research, PO BOX 17, 6700 EV Wageningen, The Netherlands e-mail: Nora.Sutton@wur.nl G. Brucha Institute of Science and Technology, Universidade Federal de Alfenas, Alfenas, Brazil P. Peng Á S. Atashgahi Á H. Smidt Laboratory of Microbiology, Wageningen University & Research, Wageningen, The Netherlands 123 Biodegradation (2021) 32:419–433 https://doi.org/10.1007/s10532-021-09940-3