Our understanding of the microorganisms involved in in situ biodegradation of xenobiotics, like pesticides, in natural and engineered environments is poor. On-farm biopurification systems (BPSs) treat farm-produced pesticide-contaminated wastewater to reduce surface water pollution. BPSs are a labor and cost-efficient technology but are still mainly operated as black box systems. We used DNA-stable isotope probing (DNA-SIP) and classical enrichment to be informed about the organisms responsible for in situ degradation of the phenylurea herbicide linuron in a BPS matrix. DNA-SIP identified Ramlibacter, Variovorax, and an unknown Comamonadaceae genus as the dominant linuron assimilators. While linuron-degrading Variovorax strains have been isolated repeatedly, Ramlibacter has never been associated before with linuron degradation. Genes and mobile genetic elements (MGEs) previously linked to linuron catabolism were enriched in the heavy DNA-SIP fractions, suggesting their involvement in in situ linuron assimilation. BPS material free cultivation of linuron degraders from the same BPS matrix resulted in a community dominated by Variovorax, while Ramlibacter was not observed. Our study provides evidence for the role of Variovorax in in situ linuron biodegradation in a BPS, alongside other organisms like Ramlibacter, and further shows that cultivation results in a biased representation of the in situ linuron-assimilating bacterial populations.

The frequent exposure of agricultural soils to pesticides often leads to microbial adaptation, including the development of dedicated microbial populations that utilize the pesticide compound as a carbon and energy source. Soil from an agricultural field in Halen (Belgium) with a history of linuron exposure has been studied for its linuron-degrading bacterial populations at two time points over the past decade and Variovorax was appointed as a key linuron degrader. Like most studies on pesticide degradation, these studies relied on isolates that were retrieved through bias-prone enrichment procedures and therefore might not represent the in situ active pesticide-degrading populations. In this study, we revisited the Halen field and applied, in addition to enrichment-based isolation, DNA stable isotope probing (DNA-SIP), to identify the in situ linuron degrading bacteria. DNA-SIP unambiguously linked Variovorax and its linuron catabolic genes to linuron dissipation, likely through synergistic cooperation between two species. Additionally, two linuron mineralizing Variovorax isolates were obtained with high 16S rRNA gene sequence similarity to strains isolated from the same field a decade earlier. The results confirm Variovorax as the in situ degrader of linuron in the studied agricultural field and corroborate the genus as key in the maintenance of a robust genetic memory regarding linuron degradation functionality in the examined field.

The frequent exposure of agricultural soils to pesticides can lead to microbial adaptation, including the development of dedicated microbial populations that utilize the pesticide compound as a carbon and energy source. Soil from an agricultural field in Halen (Belgium) with a history of linuron exposure has been studied for its linuron-degrading bacterial populations at two time points over the past decade and Variovorax was appointed as a key linuron degrader. Like most studies on pesticide degradation, these studies relied on isolates that were retrieved through bias-prone enrichment procedures and therefore might not represent the in situ active pesticide-degrading populations. In this study, we revisited the Halen field and applied, in addition to enrichment-based isolation, DNA stable isotope probing (DNA-SIP), to identify in situ linuron degrading bacteria in linuron-exposed microcosms containing that soil. Linuron dissipation was unambiguously linked to Variovorax and its linuron catabolic genes and might involve the synergistic cooperation between two species. Additionally, two novel linuron mineralizing Variovorax isolates were obtained with high 16S rRNA gene sequence similarity to strains isolated from the same field a decade earlier. The results confirm Variovorax as a prime in situ degrader of linuron in the studied agricultural field soil and corroborate the genus as key in the maintenance of the genetic memory of linuron degradation functionality in that field.