Unveiling the degradation mechanism of 3,5-dichloroaniline: Activated sludge acclimation, strain isolation and gene cloning

3,5-Dichloroaniline (3,5-DCA) is extensively used in synthesizing dicarboximide fungicides, medical compounds and dyes. Due to its widespread use in agriculture and industry, 3,5-DCA is often detected in groundwater, wastewater, sediments and soil, posing great risk to animals and humans. However, the genes and enzymes involved in 3,5-DCA degradation remain unidentified. In this study, 3,5-DCA-degrading activated sludge was obtained through 90 days of alternating anaerobic-anoxic acclimation. Microbial community analysis and further study revealed that Dehalobacter sp. DH-1 (OTU10) can convert 3,5-DCA to 3-chloroaniline anaerobically. Pseudomonas sp. DCA-1 (OTU58) can degrade 3,5-DCA, 3-chloroaniline and 2,5-dichloroaniline aerobically. Two gene clusters, ddoA1A2A3A4 and ddoBCDE , responsible for 3,5-DCA degradation, were identified via transcriptome analysis and heterologous expression. The dioxygenase genes of cluster ddoA1A2A3A4 convert 3,5-DCA to 3,5-dichlorocatechol, while those of cluster ddoBCDE mineralize the latter. Ultimately, the iprodione-degrading gene cluster ipaH-ddaH-duaH (responsible for sequential hydrolysis of the dicarboximide fungicide iprodione to 3,5-DCA) was introduced into strain DCA-1 to construct a genetically engineered microorganism, named strain IHC-DCA-1, that is capable of completely degrading iprodione. This study reveals the degradation mechanism of 3,5-DCA and provides potential strains to remediate 3,5-DCA and iprodione in the environment.