< Back to previous page

Publication

Ecology and molecular biology of bacterial adaptation to pesticides

Book - Dissertation

Pesticides are indispensable for ensuring a stable food supply chain and their world-wide use is increasing. However, they are a major environmental concern due to their recalcitrant nature and toxicity towards non-target organisms. Despite increasingly stringent regulations, pesticides continue to exceed mandated threshold levels in ground and surface waters. To address this persistence issue, a better understanding of the environmental fate of pesticides and their residues is essential. Bacterial biodegradation is an important factor governing the environmental fate of pesticides and the only route for their complete removal from the environment. The bacterial biodegradation of pesticides has been extensively studied and yet the true degradative potential in complex environments such as soil remains poorly understood. The same applies to the apparently rapid evolutionary trajectories that lead to the dedicated pathways that allow bacteria to use pesticides as growth substrate. The overall aim of this thesis was to improve our understanding of the bacterial ecology of pesticide removal and of bacterial adaptation towards pesticide degradation in soil and in on-farm biopurification systems (BPSs) used for the treatment of pesticide contaminated wastewater at farmyards. In the first part of the study, the objective was to examine whether the previous exposure of an agricultural soil to the phenylurea herbicide linuron, resulted in a genetic memory for linuron degradation within the resident bacterial community. Moreover, we examined whether linuron degraders belonging to the genus Variovorax, isolated and identified in that soil in previous studies, are instrumental for that genetic memory or mere artifacts of a biased isolation procedure. Therefore, the in situ degraders of linuron were determined for the first time by cultivation independent DNA stable isotope probing (DNA-SIP) in combination with 16S rRNA gene amplicon sequencing, in addition to classical cultivation dependent enrichment and isolation. Both approaches resulted in the assignment of Variovorax as the key linuron degrader and hence as key for maintaining the genetic memory of linuron degradation in the soil, despite periods without linuron application. The results thereby confirmed the conclusions of the previous studies and corroborated the hypothesized importance of this genus for in situ linuron removal. Nevertheless, the two novel linuron-degrading Variovorax isolates represented genotypes that were different from those identified through DNA-SIP and exemplified the potential bias of current isolation approaches. The second part of the study aimed at identifying the in situ linuron degraders in a BPS environment. As in the first part, to this end, DNA-SIP was used and in parallel, from the same material, BPS material-free enrichment cultures were studied to explore cultivation-based biases in recovering information on the linuron-degrading organisms. The DNA-SIP analysis identified, in addition to Variovorax, Ramlibacter as key in in situ linuron degradation. Orgamisms of the Ramlibacter genus were previously never associated with linuron degradation. The material-free enrichment, on the other hand, revealed vastly different degrader communities that rather resembled those previously retrieved from soil by cultivation, dominated by Variovorax but not containing Ramlibacter. This clearly showed the effect of enrichment bias. Additionally, the results showed the clear involvement of several genes previously linked to linuron catabolism in Variovorax in in situ linuron degradation, as well as their likely embedding in IS1071 composite transposons on IncP-1 plasmids. In the final part of the study, the adaptation of linuron preconditioned bacterial communities towards the degradation of the structurally similar compounds chlorpropham and chlorotoluron was explored to determine the role of a genetic memory for linuron degradation in adaptation, for instance by exchange of complementary catabolic gene modules. The linuron preconditioned BPS material studied in part two was used as the model system because of its confirmed genetic memory for linuron degradation. In addition, non-linuron preconditioned BPS material and a home-prepared BPS material never exposed to pesticides (denominated OF/O) functioned as controls. The three materials were regularly dosed with chlorpropham, chlorotoluron or pesticide-free water for up to 17 months and monitored for the mineralization of the respective pesticide, while the organisms/gene functions involved in degradation were identified by DNA-SIP and cultivation dependent enrichment. The results revealed a positive effect of linuron preconditioning for mineralization of chlorpropham and chlorotoluron. However, the observed effects were explained by an already existing ability to degrade these compounds by a subset of linuron degraders rather than by genetic exchange-mediated adaptation. This was confirmed by the delayed mineralization activity for both compounds in the OF/O material and the occurrence of the same degraders in all materials. The DNA-SIP analyses further identified previously unknown degraders for all tested compounds and suggested that the substrate range of the linuron hydrolase LibA includes chlorpropham. In conclusion, this thesis expands the existing knowledge regarding in situ pesticide biodegradation in both soil and BPS environments, particularly with regard to linuron, chlorpropham and chlorotoluron degradation. For all studied compounds, previously unknown degraders were identified by using the cultivation-independent DNA-SIP approach, while demonstrating the inherent biases associated with classical enrichment methodologies. Finally, this dissertation contributed to expanding our knowledge regarding the bacterial adaptation to pesticides and the role of genetic memory therein. This information is important for a better understanding of the environmental fate of pesticides and the underlying mechanisms of pesticide biodegradation in soil and dedicated bioremediation systems such as BPS.
Publication year:2021
Accessibility:Open