Potential and limitations for monitoring of pesticide biodegradation at trace concentrations in water and soil

Pesticides application on agricultural fields results in pesticides being released into the environment, reaching soil, surface water and groundwater. Pesticides fate and transformation in the environment depend on environmental conditions as well as physical, chemical and biological degradation processes. Monitoring pesticides biodegradation in the environment is challenging, considering that traditional indicators, such as changes in pesticides concentration or identification of pesticide metabolites, are not suitable for many pesticides in anaerobic environments. Furthermore, those indicators cannot distinguish between biotic and abiotic pesticide degradation processes. For that reason, the use of molecular tools is important to monitor pesticide biodegradation-related genes or microorganisms in the environment. The development of targeted molecular (e.g., qPCR) tools, although laborious, allowed biodegradation monitoring by targeting the presence and expression of known catabolic genes of popular pesticides. Explorative molecular tools (i.e., metagenomics & metatranscriptomics), while requiring extensive data analysis, proved to have potential for screening the biodegradation potential and activity of more than one compound at the time. The application of molecular tools developed in laboratory and validated under controlled environments, face challenges when applied in the field due to the heterogeneity in pesticides distribution as well as natural environmental differences. However, for monitoring pesticides biodegradation in the field, the use of molecular tools combined with metadata is an important tool for understanding fate and transformation of the different pesticides present in the environment

Bioremediation of pesticides: unravelling the anaerobic microbiological potential of oligotrophic drinking water aquifers

Groundwater, together with glaciers and polar icecaps, are the main reservoirs of freshwater in the world. In the Netherlands, more than half of the drinking water is being produced by using groundwater. Recent studies have shown that despite the lower exposure of the groundwater, some contaminants such as pesticides, are present in groundwater systems at micropollutant concentrations. Bioremediation is an environmentally friendly technology that relies on living organisms to biodegrade contaminants. In-situ micropollutants remediation represents a very promising but also delicate measure, because drinking water sources need to be protected from any activity that could represent a risk for drinking water production. Bioremediation processes in groundwater can be limited by the oligotrophic environmental conditions in combination with the low pesticides concentration. Bioremediation technologies that rely on pesticides biodegradation have previously been presented as a possible solution to remove pesticides from groundwater systems, making clear that more research is necessary before safe and effective application. The main goal of this thesis was to better understand the biological and environmental factors playing a role in the biodegradation of pesticides at micropollutants concentration in groundwater systems used for drinking water production in the Netherlands. The different chapters of this book contributed to: (a) create an overview of molecular tools that facilitate anaerobic pesticide monitored natural attenuation, (b) determine which groundwater microbial communities are present in the field and what are the environmental factors exerting selective pressure on them, (c) discover the metabolic potential for the biodegradation of pesticides of microbial communities in the field, (d) understand which environmental factors can facilitate pesticides biodegradation by indigenous microorganisms present in natural groundwater systems, (e) comprehend the fate of an added degrading inoculum to an stable groundwater column system. Finally, a general discussion is presented as well as some recommendations and further research lines are proposed to continue exploring the potential of microbial communities for the biodegradation of pesticides at micropollutants in groundwater systems used for drinking water production

Data underlying Biostimulation is a valuable tool to assess pesticide biodegradation capacity of groundwater microorganisms

Groundwater samples were collected at a drinking water abstraction aquifer at two locations, five different depths. Biodegradation of the MPs BAM, MCPP and 2,4-D was assessed in microcosms with groundwater samples, either without amendment, or amended with electron acceptor (nitrate or oxygen) and/or carbon substrate (dissolved organic carbon (DOC)). Oxygen + DOC was the most successful amendment resulting in complete biodegradation of 2,4-D in all microcosms after 42 days. DOC was most likely used as a growth substrate that enhanced co-metabolic 2,4-D degradation with oxygen as electron acceptor

Biostimulation is a valuable tool to assess pesticide biodegradation capacity of groundwater microorganisms

Groundwater is the main source for drinking water production globally. Groundwater unfortunately can contain micropollutants (MPs) such as pesticides and/or pesticide metabolites. Biological remediation of MPs in groundwater requires an understanding of natural biodegradation capacity and the conditions required to stimulate biodegradation activity. Thus, biostimulation experiments are a valuable tool to assess pesticide biodegradation capacity of field microorganisms. To this end, groundwater samples were collected at a drinking water abstraction aquifer at two locations, five different depths. Biodegradation of the MPs BAM, MCPP and 2,4-D was assessed in microcosms with groundwater samples, either without amendment, or amended with electron acceptor (nitrate or oxygen) and/or carbon substrate (dissolved organic carbon (DOC)). Oxygen + DOC was the most successful amendment resulting in complete biodegradation of 2,4-D in all microcosms after 42 days. DOC was most likely used as a growth substrate that enhanced co-metabolic 2,4-D degradation with oxygen as electron acceptor. Different biodegradation rates were observed per groundwater sample. Overall, microorganisms from the shallow aquifer had faster biodegradation rates than those from the deep aquifer. Higher microbial activity was also observed in terms of CO2 production in the microcosms with shallow groundwater. Our results seem to indicate that shallow groundwater contains more active microorganisms, possibly due to their exposure to higher concentrations of both DOC and MPs. Understanding field biodegradation capacity is a key step towards developing further bioremediation-based technologies. Our results show that biostimulation has real potential as a technology for remediating MPs in aquifers in order to ensure safe drinking production.</p

Exploring groundwater microbial communities for natural attenuation potential of micropollutants (pesticides)

Groundwater is a key water resource, with 45.7% of all drinking water globally being extracted from groundwater. Maintaining good groundwater quality is thus crucial to secure drinking water. Micropollutants, such as pesticides, threaten groundwater quality which can be mitigated by biodegradation. Hence, exploring microbial communities in aquifers used for drinking water production is essential for understanding micropollutants biodegradation capacity. This study aimed at understanding the interaction between groundwater geochemistry, pesticide presence, and microbial communities in aquifers used for drinking water production. Two drinking water wells located in the northeast of The Netherlands and at 500 m distance from each other were sampled in 2014, 2015, 2016 and 2018. In both wells, water was extracted from five discrete depths ranging from 13 to 54 m and used to analyze geochemical parameters, pesticide concentrations and microbial community dynamics using 16S rRNA gene sequencing and qPCR. Groundwater geochemistry was stable throughout the study period and pesticides were heterogeneously distributed at low concentrations (µg/L range). Integration of the groundwater chemical and microbial data showed that geochemical parameters and pesticides exerted selective pressure on microbial communities. Furthermore, microbial communities in both wells showed a more similar composition in the deeper part of the aquifer as compared to shallow sections, suggesting vertical differences in hydrological connection. This study provides initial insights into microbial community composition and distribution in groundwater systems in relation to geochemical parameters. This information can contribute for the implementation of bioremediation technologies that guarantee safe drinking water production from clean aquifers

2,4-Dichlorophenoxyacetic acid degradation in methanogenic mixed cultures obtained from Brazilian Amazonian soil samples

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 potential 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 degradation. In this study, methanogenic enrichment cultures were obtained from Amazonian top soil (0 - 40 cm) and deep soil (50 -80 cm below ground) that degrade 2,4-D (5 µM) to 4-chlorophenol and phenol. When 10% of these cultures were transferred to fresh medium containing 40 µM or 160 µM 2,4-D, the rate of 2,4-D degradation decreased, and the final degradation metabolites changed respectively to 4-chlorophenol and 2,4-dichlorophenol in the top and deep soil cultures. 16S rRNA community diversity analysis 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 the biodegradation pathway and microbial diversity, and these are both important factors when developing pesticide bioremediation technologies

Selective pressure on microbial communities in a drinking water aquifer – Geochemical parameters vs. micropollutants

Groundwater quality is crucial for drinking water production, but groundwater resources are increasingly threatened by contamination with pesticides. As pesticides often occur at micropollutant concentrations, they are unattractive carbon sources for microorganisms and typically remain recalcitrant. Exploring microbial communities in aquifers used for drinking water production is an essential first step towards understanding the fate of micropollutants in groundwater. In this study, we investigated the interaction between groundwater geochemistry, pesticide presence, and microbial communities in an aquifer used for drinking water production. Two groundwater monitoring wells in The Netherlands were sampled in 2014, 2015, and 2016. In both wells, water was sampled from five discrete depths ranging from 13 to 54 m and was analyzed for geochemical parameters, pesticide concentrations and microbial community composition using 16S rRNA gene sequencing and qPCR. Groundwater geochemistry was stable throughout the study period and pesticides were heterogeneously distributed at low concentrations (μg L−1 range). Microbial community composition was also stable throughout the sampling period. Integration of a unique dataset of chemical and microbial data showed that geochemical parameters and to a lesser extent pesticides exerted selective pressure on microbial communities. Microbial communities in both wells showed similar composition in the deeper aquifer, where pumping results in horizontal flow. This study provides insight into groundwater parameters that shape microbial community composition. This information can contribute to the future implementation of remediation technologies to guarantee safe drinking water production

2,4-Dichlorophenoxyacetic acid degradation in methanogenic mixed cultures obtained from Brazilian Amazonian soil samples

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, 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.</p