Shedding light on the total and active core microbiomes in slow sand filters for drinking water production.

Slow sand filters (SSF) are widely used in the production of drinking water as a last barrier in the removal of pathogens. This removal mainly depends on the 'Schmutzdecke', a biofilm-like layer on the surface of the sand bed. Most previous studies focused on the total community as revealed by DNA analysis rather than on the active community, which may lead to an incorrect understanding of the SSF ecology. In the current study, we determined and compared the DNA- (total) and RNA-displayed (active) communities in the Schmutzdecke layer from 10 full-scale slow sand filters and further explored the SSF core microbiome in terms of both presence (DNA) and activity (RNA). Discrepancies were observed between the total and the active community, although there was a consistent grouping in the PCoA analysis. The DNA-displayed community may be somewhat inflated, while the RNA-displayed community could reveal low abundance (or rare) but active community members. The overall results imply that both DNA (presence) and RNA (activity) data should be considered to prevent the underestimation of organisms of functional importance but lower abundance. Microbial communities of studied mature Schmutzdecke were shaped by the influent water. Nevertheless, a core microbiome was shared by the mature Schmutzdeckes from independent filters, representing the dominant and consistent microbial community composition in slow sand filters. In the DNA samples, a total of 33 VSC families ('very strict core', with a relative abundance >0.1% and 100% prevalence) were observed across all filters. Among the RNA samples, there were 18 VSC families, including 16 families that overlapped with the DNA VSC families and 2 unique RNA VSC families. The core microbial community structure was influenced by the operational parameters, including the Schmutzdecke age and the sand size, and was less influenced by water flow. In addition, indicator organisms ('biomarkers') for the Schmutzdecke age, which show the longest duration that SSF can maintain a good operation, were observed in our study. The abundant presence of bacteria belonging to bacteriap25 and Caldilineaceae was associated with older Schmutzdeckes, revealing longer periods of stable operation performance of the filter, while the high abundance of bacteria belonging to Bdellovibrionaceae and Bryobacteraceae related to short periods of stable operation performance.

A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool.

Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine application in environmental quality monitoring, an easy-to-use multiplex assay is required to reliably quantify expression levels of these biomarkers. In the present study, a bead-based assay was developed to fingerprint gene expression in C. elegans by quantitating messenger RNAs (mRNAs) of multiple target genes directly from crude nematode lysates, circumventing RNA extraction and purification steps. The assay uses signal amplification rather than target amplification for direct measurement of toxin-induced RNA transcripts. Using a 50-gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time-consuming reverse-transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead-based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130-142. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Determining Toxic Potencies of Water-Soluble Contaminants in Wastewater Influents and Effluent Using Gene Expression Profiling in C. elegans as a Bioanalytical Tool.

With chemical analysis, it is impossible to qualify and quantify the toxic potency of especially hydrophilic bioactive contaminants. In this study, we applied the nematode C. elegans as a model organism for detecting the toxic potency of whole influent wastewater samples. Gene expression in the nematode was used as bioanalytical tool to reveal the presence, type and potency of molecular pathways induced by 24-h exposure to wastewater from a hospital (H), nursing home (N), community (C), and influent (I) and treated effluent (E) from a local wastewater treatment plant. Exposure to influent water significantly altered expression of 464 genes, while only two genes were differentially expressed in nematodes treated with effluent. This indicates a significant decrease in bioactive pollutant-load after wastewater treatment. Surface water receiving the effluent did not induce any genes in exposed nematodes. A subset of 209 genes was differentially expressed in all untreated wastewaters, including cytochromes P450 and C-type lectins related to the nematode's xenobiotic metabolism and immune response, respectively. Different subsets of genes responded to particular waste streams making them candidates to fingerprint-specific wastewater sources. This study shows that gene expression profiling in C. elegans can be used for mechanism-based identification of hydrophilic bioactive compounds and fingerprinting of specific wastewaters. More comprehensive than with chemical analysis, it can demonstrate the effective overall removal of bioactive compounds through wastewater treatment. This bioanalytical tool can also be applied in the process of identification of the bioactive compounds via a process of toxicity identification evaluation.

Microbial ecology of biofiltration used for producing safe drinking water.

Biofiltration is a water purification technology playing a pivotal role in producing safe drinking water. This technology attracts many interests worldwide due to its advantages, such as no addition of chemicals, a low energy input, and a high removal efficiency of organic compounds, undesirable taste and odours, and pathogens. The current review describes the microbial ecology of three biofiltration processes that are routinely used in drinking water treatment plants, i.e. (i) rapid sand filtration (RSF), (ii) granular activated carbon filtration (GACF), and (iii) slow sand filtration (SSF). We summarised and compared the characteristics, removal performance, and corresponding (newly revealed) mechanisms of the three biofiltration processes. Specifically, the microbial ecology of the different biofilter processes and the role of microbial communities in removing nutrients, organic compounds, and pathogens were reviewed. Finally, we highlight the limitations and challenges in the study of biofiltration in drinking water production, and propose future perspectives for obtaining a comprehensive understanding of the microbial ecology of biofiltration, which is needed to promote and optimise its further application. KEY POINTS: • Biofilters are composed of complex microbiomes, primarily shaped by water quality. • Conventional biofilters contribute to address safety challenges in drinking water. • Studies may underestimate the active/functional role of microbiomes in biofilters.

Clean and Safe Drinking Water Systems via Metagenomics Data and Artificial Intelligence: State-of-the-Art and Future Perspective.

The use of next-generation sequencing technologies in drinking water distribution systems (DWDS) has shed insight into the microbial communities' composition, and interaction in the drinking water microbiome. For the past two decades, various studies have been conducted in which metagenomics data have been collected over extended periods and analyzed spatially and temporally to understand the dynamics of microbial communities in DWDS. In this literature review, we outline the findings which were reported in the literature on what kind of occupancy-abundance patterns are exhibited in the drinking water microbiome, how the drinking water microbiome dynamically evolves spatially and temporally in the distribution networks, how different microbial communities co-exist, and what kind of clusters exist in the drinking water ecosystem. While data analysis in the current literature concerns mainly with confirmatory and exploratory questions pertaining to the use of metagenomics data for the analysis of DWDS microbiome, we present also future perspectives and the potential role of artificial intelligence (AI) and mechanistic models to address the predictive and mechanistic questions. The integration of meta-omics, AI, and mechanistic models transcends metagenomics into functional metagenomics, enabling deterministic understanding and control of DWDS for clean and safe drinking water systems of the future.

Differential expression of genes in C. elegans reveals transcriptional responses to indirect-acting xenobiotic compounds and insensitivity to 2,3,7,8-tetrachlorodibenzodioxin.

Caenorhabditis elegans is a well-established model organism for toxicity testing of chemical substances. We recently demonstrated its potential for bioanalysis of the toxic potency of chemical contaminants in water. While many detoxification genes are homologues to those in mammalians, C. elegans is reported to be deficient in cytochrome CYP1-like P450 metabolism and that its aryl hydrocarbon receptor (AhR) homolog encoded by ahr-1 purportedly does not interact with dioxins or any other known xenobiotic ligand. This suggests that C. elegans is insensitive for compounds that require bioactivation (indirectly acting compounds) and for dioxins or dioxin-like compounds. This study analysed genome-wide gene expression of the nematode in response to 30 µM of aflatoxin B1 (AFB1), benzo(a)pyrene (B(a)P), Aroclor 1254 (PCB1254), and 10 µM of 2,3,7,8-tetrachlorodibenzodioxin (TCDD). After 24 h of exposure in the early L4 larval stage, microarray analysis revealed 182, 86, and 321 differentially expressed genes in the nematodes treated with 30 µM of AFB1, B(a)P, and PCB1254, respectively. Among these genes, many encode xenobiotic-metabolizing enzymes, and their transcription levels were among the highest-ranked fold-changed genes. Interestingly, only one gene (F59B1.8) was upregulated in the nematodes exposed to 10 µM TCDD. Genes related to metabolic processes and catalytic activity were the most induced by exposure to 30 µM of AFB1, B(a)P, and PCB1254. Despite the genotoxic nature of AFB1 and B(a)P, no differential expression was found in the genes encoding DNA repair and cell cycle checkpoint proteins. Analysis of concentration-response curves was performed to determine the Lowest Observed Transcriptomic Effect Levels (LOTEL) of AFB1, B(a)P, and PCB1254. The obtained LOTEL values showed that gene expression changes in C. elegans are more sensitive to toxicants than reproductive effects. Overall, transcriptional responses of metabolic enzymes suggest that the nematode does metabolize AFB1, B(a)P, and PCB1254. Our findings also support the assumption that the transcription factor AhR homolog in C. elegans does not bind typical xenobiotic ligands, rendering the nematode transcriptionally insensitive to TCDD effects.

Development of a transcription-based bioanalytical tool to quantify the toxic potencies of hydrophilic compounds in water using the nematode Caenorhabditis elegans.

Low concentrations of environmental contaminants can be difficult to detect with current analytical tools, yet they may pose a risk to human and environmental health. The development of bioanalytical tools can help to quantify toxic potencies of biologically active compounds even of hydrophilic contaminants that are hard to extract from water samples. In this study, we exposed the model organism Caenorhabditis elegans synchronized in larval stage L4 to hydrophilic compounds via the water phase and analyzed the effect on gene transcription abundance. The nematodes were exposed to three direct-acting genotoxicants (1 mM and 5 mM): N-ethyl-N-nitrosourea (ENU), formaldehyde (HCHO), and methyl methanesulfonate (MMS). Genome-wide gene expression analysis using microarrays revealed significantly altered transcription levels of 495 genes for HCHO, 285 genes for ENU, and 569 genes for MMS in a concentration-dependent manner. A relatively high number of differentially expressed genes was downregulated, suggesting a general stress in nematodes treated with toxicants. Gene ontology and Kyoto encyclopedia of genes and genomes analysis demonstrated that the upregulated genes were primarily associated with metabolism, xenobiotic detoxification, proteotoxic stress, and innate immune response. Interestingly, genes downregulated by MMS were linked to the inhibition of neurotransmission, and this is in accordance with the observed decreased locomotion in MMS-exposed nematodes. Unexpectedly, the expression level of DNA damage response genes such as cell-cycle checkpoints or DNA-repair proteins were not altered. Overall, the current study shows that gene expression profiling of nematodes can be used to identify the potential mechanisms underlying the toxicity of chemical compounds. C. elegans is a promising test organism to further develop into a bioanalytical tool for quantification of the toxic potency of a wide array of hydrophilic contaminants.

Density-dependent microbial calcium carbonate precipitation by drinking water bacteria via amino acid metabolism and biosorption.

Drinking water plumbing systems appear to be a unique environment for microorganisms as they contain few nutrients but a high mineral concentration. Interactions between mineral content and bacteria, such as microbial calcium carbonate precipitation (MCP) however, has not yet attracted too much attention in drinking water sector. This study aims to carefully examine MCP behavior of two drinking water bacteria species, which may potentially link scaling and biofouling processes in drinking water distribution systems. Evidence from cell density evolution, chemical parameters, and microscopy suggest that drinking water isolates can mediate CaCO3 precipitation through previously overlooked MCP mechanisms like ammonification or biosorption. The results also illustrate the active control of bacteria on the MCP process, as the calcium starts to concentrate onto cell surfaces only after reaching a certain cell density, even though the cell surfaces are shown to be the ideal location for the CaCO3 nucleation.

Integration of biopolymer production with process water treatment at a sugar factory.

The present investigation has focused on generating a surplus denitrifying biomass with high polyhydroxyalkanoate (PHA) producing potential while maintaining water treatment performance in biological nitrogen removal. The motivation for the study was to examine integration of PHA production into the water treatment and residuals management needs at the Suiker Unie sugar beet factory in Groningen, the Netherlands. At the factory, process waters are treated in nitrifying-denitrifying sequencing batch reactors (SBRs) to remove nitrogen found in condensate. Organic slippage (COD) in waters coming from beet washing is the substrate used for denitrification. The full-scale SBR was mimicked at laboratory scale. In two parallel laboratory scale SBRs, a mixed-culture biomass selection strategy of anoxic-feast and aerobic-famine was investigated using the condensate and wash water from Suiker Unie. One laboratory SBR was operated as conventional activated sludge with long solids retention time similar to the full-scale (SRT >16 days) while the other SBR was a hybrid biofilm-activated sludge (IFAS) process with short SRT (4-6 days) for the suspended solids. Both SBRs were found to produce biomass with augmented PHA production potential while sustaining process water treatment for carbon, nitrogen and phosphorus for the factory process waters. PHA producing potential in excess of 60 percent g-PHA/g-VSS was achieved with the lab scale surplus biomass. Surplus biomass of low (4-6 days) and high (>16 days) solids retention time yielded similar results in PHA accumulation potential. However, nitrification performance was found to be more robust for the IFAS SBR. Assessment of the SBR microbial ecology based on 16sDNA and selected PHA synthase genes at full-scale in comparison to biomass from the laboratory scale SBRs suggested that the full-scale process was enriched with a PHA storing microbial community. However, structure-function relationships based on RNA levels for the selected PHA synthases could not be established and, towards this ambition, it is speculated that a wider representation of PHA synthesases would need to be monitored. Additionally at the factory, beet tail press waters coming from the factory beet residuals management activities are available as a carbon source for PHA accumulation. At pilot scale, beet tail press waters were shown to provide a suitable carbon source for mixed culture PHA production in spite of otherwise being of relatively low organic strength (≤ 10 g-COD/L). A copolymer of 3-hydroxybutyrate with 3-hydroxyvalerate (PHBV with 15% HV on a molar basis) of high thermal stability and high weight average molecular mass (980 kDa) was produced from the beet tail press water. The mixed culture accumulation process sustained PHA storage with parallel biomass growth of PHA storing bacteria suggesting a strategy to further leverage the utilization of surplus functional biomass from biological treatment systems. Integration of PHA production into the existing factory water management by using surplus biomass from condensate water treatment and press waters from beet residuals processing was found to be a feasible strategy for biopolymer production.

Ecogenomics of microbial communities in bioremediation of chlorinated contaminated sites.

Organohalide compounds such as chloroethenes, chloroethanes, and polychlorinated benzenes are among the most significant pollutants in the world. These compounds are often found in contamination plumes with other pollutants such as solvents, pesticides, and petroleum derivatives. Microbial bioremediation of contaminated sites, has become commonplace whereby key processes involved in bioremediation include anaerobic degradation and transformation of these organohalides by organohalide respiring bacteria and also via hydrolytic, oxygenic, and reductive mechanisms by aerobic bacteria. Microbial ecogenomics has enabled us to not only study the microbiology involved in these complex processes but also develop tools to better monitor and assess these sites during bioremediation. Microbial ecogenomics have capitalized on recent advances in high-throughput and -output genomics technologies in combination with microbial physiology studies to address these complex bioremediation problems at a system level. Advances in environmental metagenomics, transcriptomics, and proteomics have provided insights into key genes and their regulation in the environment. They have also given us clues into microbial community structures, dynamics, and functions at contaminated sites. These techniques have not only aided us in understanding the lifestyles of common organohalide respirers, for example Dehalococcoides, Dehalobacter, and Desulfitobacterium, but also provided insights into novel and yet uncultured microorganisms found in organohalide respiring consortia. In this paper, we look at how ecogenomic studies have aided us to understand the microbial structures and functions in response to environmental stimuli such as the presence of chlorinated pollutants.

Population dynamics of a single-stage sulfidogenic bioreactor treating synthetic zinc-containing waste streams.

Waste streams from industrial processes such as metal smelting or mining contain high concentrations of sulfate and metals with low pH. Dissimilatory sulfate reduction carried out by sulfate-reducing bacteria (SRB) at low pH can combine sulfate reduction with metal-sulfide precipitation and thus open possibilities for selective metal recovery. This study investigates the microbial diversity and population changes of a single-stage sulfidogenic gas-lift bioreactor treating synthetic zinc-rich waste water at pH 5.5 by denaturing gradient gel electrophoresis of 16S rRNA gene fragments and quantitative polymerase chain reaction. The results indicate the presence of a diverse range of phylogenetic groups with the predominant microbial populations belonging to the Desulfovibrionaceae from delta-Proteobacteria. Desulfovibrio desulfuricans-like populations were the most abundant among the SRB during the three stable phases of varying sulfide and zinc concentrations and increased from 13% to 54% of the total bacterial populations over time. The second largest group was Desulfovibrio marrakechensis-like SRB that increased from 1% to about 10% with decreasing sulfide concentrations. Desulfovibrio aminophilus-like populations were the only SRB to decrease in numbers with decreasing sulfide concentrations. However, their population was <1% of the total bacterial population in the reactor at all analyzed time points. The number of dissimilatory sulfate reductase (DsrA) gene copies per number of SRB cells decreased from 3.5 to 2 DsrA copies when the sulfide concentration was reduced, suggesting that the cells' sulfate-reducing capacity was also lowered. This study has identified the species present in a single-stage sulfidogenic bioreactor treating zinc-rich wastewater at low pH and provides insights into the microbial ecology of this biotechnological process.

Bacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme activities.

Bacterial dehalogenases catalyse the cleavage of carbon-halogen bonds, which is a key step in aerobic mineralization pathways of many halogenated compounds that occur as environmental pollutants. There is a broad range of dehalogenases, which can be classified in different protein superfamilies and have fundamentally different catalytic mechanisms. Identical dehalogenases have repeatedly been detected in organisms that were isolated at different geographical locations, indicating that only a restricted number of sequences are used for a certain dehalogenation reaction in organohalogen-utilizing organisms. At the same time, massive random sequencing of environmental DNA, and microbial genome sequencing projects have shown that there is a large diversity of dehalogenase sequences that is not employed by known catabolic pathways. The corresponding proteins may have novel functions and selectivities that could be valuable for biotransformations in the future. Apparently, traditional enrichment and metagenome approaches explore different segments of sequence space. This is also observed with alkane hydroxylases, a category of proteins that can be detected on basis of conserved sequence motifs and for which a large number of sequences has been found in isolated bacterial cultures and genomic databases. It is likely that ongoing genetic adaptation, with the recruitment of silent sequences into functional catabolic routes and evolution of substrate range by mutations in structural genes, will further enhance the catabolic potential of bacteria toward synthetic organohalogens and ultimately contribute to cleansing the environment of these toxic and recalcitrant chemicals.