Integrating taxonomic signals from MAGs and contigs improves read annotation and taxonomic profiling of metagenomes.

Metagenomic analysis typically includes read-based taxonomic profiling, assembly, and binning of metagenome-assembled genomes (MAGs). Here we integrate these steps in Read Annotation Tool (RAT), which uses robust taxonomic signals from MAGs and contigs to enhance read annotation. RAT reconstructs taxonomic profiles with high precision and sensitivity, outperforming other state-of-the-art tools. In high-diversity groundwater samples, RAT annotates a large fraction of the metagenomic reads, calling novel taxa at the appropriate, sometimes high taxonomic ranks. Thus, RAT integrative profiling provides an accurate and comprehensive view of the microbiome from shotgun metagenomics data. The package of Contig Annotation Tool (CAT), Bin Annotation Tool (BAT), and RAT is available at https://github.com/MGXlab/CAT_pack (from CAT pack v6.0). The CAT pack now also supports Genome Taxonomy Database (GTDB) annotations.

Bioaugmentation has temporary effect on anaerobic pesticide biodegradation in simulated groundwater systems.

Groundwater is the most important source for drinking water in The Netherlands. Groundwater quality is threatened by the presence of pesticides, and biodegradation is a natural process that can contribute to pesticide removal. Groundwater conditions are oligotrophic and thus biodegradation can be limited by the presence and development of microbial communities capable of biodegrading pesticides. For that reason, bioremediation technologies such as bioaugmentation (BA) can help to enhance pesticide biodegradation. We studied the effect of BA using enriched mixed inocula in two column bioreactors that simulate groundwater systems at naturally occurring redox conditions (iron and sulfate-reducing conditions). Columns were operated for around 800 days, and two BA inoculations (BA1 and BA2) were conducted in each column. Inocula were enriched from different wastewater treatment plants (WWTPs) under different redox-conditions. We observed a temporary effect of BA1, reaching 100% removal efficiency of the pesticide 2,4-D after 100 days in both columns. In the iron-reducing column, 2,4-D removal was in general higher than under sulfate-reducing conditions demonstrating the influence of redox conditions on overall biodegradation. We observed a temporary shift in microbial communities after BA1 that is relatable to the increase in 2,4-D removal efficiency. After BA2 under sulfate-reducing conditions, 2,4-D removal efficiency decreased, but no change in the column microbial communities was observed. The present study demonstrates that BA with a mixed inoculum can be a valuable technique for improving biodegradation in anoxic groundwater systems at different redox-conditions.

Indene, indane and naphthalene in a mixture with BTEX affect aerobic compound biodegradation kinetics and indigenous microbial community development.

BTEX (benzene, toluene, ethylbenzene, xylene) are common pollutants often found in former gasworks sites together with some other contaminants like indene, indane and naphthalene (Ie, Ia, N). This study aimed to evaluate the inhibitory or stimulative substrate interactions between BTEX, and Ie, Ia, N during aerobic biodegradation. For this, batch bottles, containing originally anaerobic subsurface sediments, groundwater and indigenous microorganisms from a contaminated former gasworks site, were spiked with various substrate combinations (BTEX, BTEXIe, BTEXIa, BTEXN, BTEXIeIa, BTEXIeN, BTEXIaN, BTEXIeIaN). Subsequently concentrations were monitored over time. For the BTEXIeIaN mixture, initial concentrations were between 1 and 5 mg L-1, and all compounds were completely degraded by the microbial consortia within 39 days of incubation. The experimental data were fitted to a first order kinetic degradation model for interpretation of inhibition/stimulation between the compounds. Results showed that indene, indane, and naphthalene inhibited the degradation of benzene, toluene, ethylbenzene, o-xylene, with benzene being the most affected. M/p-xylene is the only compound whose biodegradation is stimulated by the presence of indene and indane (individually or mixed) but inhibited by the presence of naphthalene. 16S rRNA amplicon sequencing revealed differentiation in the microbial communities within the batches with different substrate mixtures, especially within the two microbial groups Micrococcaceae and Commamonaceae. Indene had more effect on the BTEX microbial community than indane or naphthalene and the presence of indene increased the relative abundance of Micrococcaceae family. In conclusion, co-presence of various pollutants leads to differentiation in degradation processes as well as in microbial community development. This sheds some light on the underlying reasons for that organic compounds present in mixtures in the subsurface of former gasworks sites are either recalcitrant or subjective towards biodegradation, and this understanding helps to further improve the bioremediation of such sites.

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.

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.

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.

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.