Benthic Diatom Communities in an Alpine River Impacted by Waste Water Treatment Effluents as Revealed Using DNA Metabarcoding

Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities’ development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 185-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data. (C) 2020 The Authors. Published by Elsevier B.V

Metadata standards and practical guidelines for specimen and DNA curation when building barcode reference libraries for aquatic life

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data.info:eu-repo/semantics/publishedVersio

Revisiting global biogeography of freshwater diatoms: new insights from molecular data

The high-dispersal rates of microorganisms have driven to the expectation of their cosmopolitan geographic distribution. However, recent studies demonstrate that microorganisms instead show particular biogeography. Despite the existence of cosmopolitan species, geographically limited microbial groups have been found in aquatic and terrestrial environments worldwide.Diatoms are long time used model to study the biogeography of microorganisms. They are unicellular eukaryotic microalgae that contribute significantly to the aquatic primary production and have huge taxonomic diversity and marked species-specific ecological preferences. Several authors considered that diatoms have no limits in dispersion and are ubiquitously present. On the other hand, recent studies have shown that endemism exists for several genera, and species may have low dispersal capacity. However, all these studies are based on data obtained by microscopy and therefore suffer from the many well-identified biases associated with the optical identification of microorganisms at large scale.Metabarcoding technologies provide an access to taxonomic precision with a higher resolution compared to microscopy and open therefore the possibility of analyzing microbial diversity at genetic level. Recent bioinformatics tools allow reliable and standardized comparison of large datasets originating from distant geographic regions, overcoming issues related to biases in species identification.In this study we used metabarcoding data to revisit central questions in freshwater diatom biogeography. We assembled a large dataset of samples of benthic diatoms collected from rivers in seven different geographic regions. These regions cover the subpolar (Fennoscandia), temperate (France Mainland) and tropical (West Africa, French Guyana, New Caledonia, Tahiti island and Mayotte island) climate zones. The selected geographic regions can also be classified into four continental areas (Fennoscandia, France Mainland, West Africa, and French Guyana) and three islands (New Caledonia, Tahiti and Mayotte).We analyzed diatom alpha, beta and gamma diversity patterns in this dataset to address two main questions: 1) the presence of a latitudinal gradient in diatom diversity and 2) the cosmopolitanism of diatoms.Similarly to results previously reported by Soininen et al. 2016, our data showed a decrease in diatom richness with a decrease in latitude. However, testing the effect of land type (island vs. mainland) showed that this factor explains the actual variability of richness along the climatic gradient and the effect of latitude is not significant. Differences in community structure between regions and climate zones were significant. In multivariate analysis, tropical samples did not overlap with any of the other climate zones, suggesting the specificity of these communities

Environmental filtering and mass effect are two important processes driving lake benthic diatoms: Results of a DNA metabarcoding study in a large lake

International audienceEnvironmental filtering is often found to dominate assembly rules in diatoms. These microalgae are diverse, especially at subspecies level, and tend to exhibit a niche phylogenetic conservatism. Therefore, other rules, such as competition or mass effects, should be detectable when environmental gradients and dispersal barriers are limited. We used metabarcoding to analyse benthic littoral diatom communities in 153 sites in a large lake (Geneva) exhibiting weak geographical barriers and weak environmental gradients outside river estuaries. We assessed assembly rules using variance partitioning, phylogenetic and source tracking analyses. No phylogenetic over-dispersion of communities, indicative of exclusive competition, was detected. Instead, we found these communities to be phylogenetically over-clustered, indicating environmental filtering, which was even stronger near river estuaries where environmental gradients are stronger. Finally, using a Bayesian method (SourceTracker), we found that rivers flowing into the lake bring communities that settle, especially in sites close to estuaries. Rivers with the highest discharges are primarily responsible for immigration, explaining 27% of lake composition. Therefore, despite favourable conditions to observe other rules, our results support that diatom communities are prominently assembled by environmental filtering and immigration processes, in particular from rivers. However, this does not exclude that other assembly rules may be at play at a finer spatial, temporal and/or phylogenetic scale

Separate treatment of hospital and urban wastewaters: A real scale comparison of effluents and their effect on microbial communities

International audienceWe compared treatment with activated sludge of hospital and urban wastewaters.•Pharmaceuticals had higher removal efficiency during hospital wastewater treatment.•Treated hospital effluents still contained higher concentrations of pharmaceuticals.•Biofilms developed in the two treated effluents had different community structure.•Biofilms in hospital treated effluents were less developed and had lower diversity

Two-year survey of specific hospital wastewater treatment and its impact on pharmaceutical discharges

International audienceIt is well known that pharmaceuticals are not completely removed by conventional activated sludge wastewater treatment plants. Hospital effluents are of major concern, as they present high concentrations of pharmaceutically active compounds. Despite this, these specific effluents are usually co-treated with domestic wastewaters. Separate treatment has been recommended. However, there is a lack of information concerning the efficiency of separate hospital wastewater treatment by activated sludge, especially on the removal of pharmaceuticals. In this context, this article presents the results of a 2-year monitoring of conventional parameters, surfactants, gadolinium, and 13 pharmaceuticals on the specific study site SIPIBEL. This site allows the characterization of urban and hospital wastewaters and their separate treatment using the same process. Flow proportional sampling, solid-phase extraction, and liquid chromatography coupled with tandem mass spectrometry were used in order to obtain accurate data and limits of quantification consistent with ultra-trace detection. Thanks to these consolidated data, an in-depth characterization of urban and hospital wastewaters was realized, as well as a comparison of treatment efficiency between both effluents. Higher concentrations of organic carbon, AOX, phosphates, gadolinium, paracetamol, ketoprofen, and antibiotics were observed in hospital wastewaters compared to urban wastewaters. Globally higher removals were observed in the hospital wastewater treatment plant, and some parameters were shown to be of high importance regarding removal efficiencies: hydraulic retention time, redox conditions, and ambient temperature. Eleven pharmaceuticals were still quantified at relevant concentrations in hospital and urban wastewaters after treatment (e.g., up to 1 μg/L for sulfamethoxazole). However, as the urban flow was about 37 times higher than the hospital flow, the hospital contribution appeared relatively low compared to domestic discharges. Thanks to the SIPIBEL site, data obtained from this 2-year program are useful to evaluate the relevance of separate hospital wastewater treatment

Same same, but different: The response of diatoms to environmental gradients in Fennoscandian streams and lakes – barcodes, traits and microscope data compared

We developed and compared the performance of freshwater benthic diatom indices calculated from (i) traditional morphological species identification, (ii) Amplicon Sequence Variants (ASVs) obtained via DNA metabarcoding, and (iii) morphological traits to indicate eutrophication in rivers and lakes in Fennoscandia. Based on the results, we provided recommendations for the future routine use of diatom bioassessment tools in environmental monitoring and assessment. Our results show that ASVs are the most promising candidates to be used in environmental assessment. Indices based on ASVs correlated better with TotP concentrations than morphological taxa data, whereas the trait indices correlated least. We could see by studying the taxonomic assignments of the ASVs that traditional morphotaxa were divided up into several ASVs with different ecological profiles, which explained part of the better index performance and also encourages further studies on diatom diversity and ecological preferences. In general, ASV- and morphotaxon-specific optima differed slightly between streams and lakes, but were significantly correlated with each other. This means that it should be possible to develop a common index that is applicable in both streams and lakes, but boundary values with respect to TotP might need to be set separately for them. More knowledge on diatom traits is required to enable their use for environmental assessment

Same same, but different : The response of diatoms to environmental gradients in Fennoscandian streams and lakes – barcodes, traits and microscope data compared

Highlights • Amplicon Sequence Variants (ASVs) are a promising unit for diatom nutrient indices. • Diatom indices based on morphology, ASVs and traits were related to TotP concentrations. • Correlations of indices to TotP was best for ASVs, followed by morphology and traits.We developed and compared the performance of freshwater benthic diatom indices calculated from (i) traditional morphological species identification, (ii) Amplicon Sequence Variants (ASVs) obtained via DNA metabarcoding, and (iii) morphological traits to indicate eutrophication in rivers and lakes in Fennoscandia. Based on the results, we provided recommendations for the future routine use of diatom bioassessment tools in environmental monitoring and assessment. Our results show that ASVs are the most promising candidates to be used in environmental assessment. Indices based on ASVs correlated better with TotP concentrations than morphological taxa data, whereas the trait indices correlated least. We could see by studying the taxonomic assignments of the ASVs that traditional morphotaxa were divided up into several ASVs with different ecological profiles, which explained part of the better index performance and also encourages further studies on diatom diversity and ecological preferences. In general, ASV- and morphotaxon-specific optima differed slightly between streams and lakes, but were significantly correlated with each other. This means that it should be possible to develop a common index that is applicable in both streams and lakes, but boundary values with respect to TotP might need to be set separately for them. More knowledge on diatom traits is required to enable their use for environmental assessment

Evidence of Bacterial Community Coalescence between Freshwater and Discharged tpm-Harboring Bacterial Taxa from Hospital and Domestic Wastewater Treatment Plants among Epilithic Biofilms

The ability of WWTP outflow bacteria at colonizing rock surfaces and contributing to the formation of river epilithic biofilms was investigated. Bacterial community structures of biofilms (b-) developing on rocks exposed to treated wastewaters (TWW) of a hospital (HTWW) and a domestic (DTWW) clarifier, and to surface waters of the stream located at 10 m, 500 m, and 8 km from the WWTP outlet, were compared. Biofilm bacterial contents were analyzed by cultural approaches and a tpm-based DNA metabarcoding analytical scheme. Co-occurrence distribution pattern analyses between bacterial datasets and eighteen monitored pharmaceuticals were performed. Higher concentrations of iohexol, ranitidine, levofloxacin, and roxithromycin were observed in the b-HTWW while atenolol, diclofenac, propranolol, and trimethoprim were higher in the b-DTWW. MPN growth assays showed recurrent occurrences of Pseudomonas aeruginosa and Aeromonas caviae among these biofilms. An enrichment of multi-resistant P. aeruginosa cells was observed in the hospital sewer line. P. aeruginosa MPN values were negatively correlated to roxithromycin concentrations. The tpm DNA metabarcoding analyses confirmed these trends and allowed an additional tracking of more than 90 species from 24 genera. Among the recorded 3082 tpm ASV (amplicon sequence variants), 41% were allocated to the Pseudomonas. Significant differences through ANOSIM and DESeq2 statistical tests were observed between ASV recovered from b-HTWW, b-DTWW, and epilithic river biofilms. More than 500 ASV were found restricted to a single sewer line such as those allocated to Aeromonas popoffii and Stenotrophomonas humi being strictly found in the b-HTWW file. Several significant correlations between tpm ASV counts per species and pharmaceutical concentrations in biofilms were recorded such as those of Lamprocystis purpurea being positively correlated with trimethoprim concentrations. A tpm source tracking analysis showed the b-DTWW and b-HTWW tpm ASV to have contributed, respectively, at up to 35% and 2.5% of the epilithic river biofilm tpm-taxa recovered downstream from the WWTP outlet. Higher contributions of TWW taxa among epilithic biofilms were recorded closer to the WWTP outlet. These analyses demonstrated a coalescence of WWTP sewer communities with river freshwater taxa among epilithic biofilms developing downstream of a WWTP outlet