Unraveling the genetic potential of nitrous oxide reduction in wastewater treatment: insights from metagenome-assembled genomes.

This study explores the genetic landscape of nitrous oxide (N2O) reduction in wastewater treatment plants (WWTPs) by profiling 1,083 high-quality metagenome-assembled genomes (HQ MAGs) from 23 Danish full-scale WWTPs. The focus is on the distribution and diversity of nitrous oxide reductase (nosZ) genes and their association with other nitrogen metabolism pathways. A custom pipeline for clade-specific nosZ gene identification with higher sensitivity revealed 503 nosZ sequences in 489 of these HQ MAGs, outperforming existing Kyoto Encyclopedia of Genes and Genomes (KEGG) module-based methods. Notably, 48.7% of the total 1,083 HQ MAGs harbored nosZ genes, with clade II being predominant, accounting for 93.7% of these genes. Taxonomic profiling highlighted the prevalence of nosZ-containing taxa within Bacteroidota and Pseudomonadota. Chloroflexota exhibited unexpected affiliations with both the sec and tat secretory pathways, and all were found to contain the accessory nosB gene, underscoring the importance of investigating the secretory pathway. The majority of non-denitrifying N2O reducers were found within Bacteroidota and Chloroflexota. Additionally, HQ MAGs with genes for dissimilatory nitrate reduction to ammonium and assimilatory nitrate reduction frequently co-occurred with the nosZ gene. Traditional primers targeting nosZ often focus on short-length amplicons. Therefore, we introduced custom-designed primer sets targeting near-full-length nosZ sequences. These new primers demonstrate efficacy in capturing diverse and well-characterized sequences, providing a valuable tool with higher resolution for future research. In conclusion, this comprehensive analysis enhances our understanding of N2O-reducing organisms in WWTPs, highlighting their potential as N2O sinks with the potential for optimizing wastewater treatment processes and mitigating greenhouse gas emissions. IMPORTANCE: This study provides critical insights into the genetic diversity of nitrous oxide reductase (nosZ) genes and the microorganisms harboring them in wastewater treatment plants (WWTPs) by exploring 1,083 high-quality metagenome-assembled genomes (MAGs) from 23 Danish full-scale WWTPs. Despite the pivotal role of nosZ-containing organisms, their diversity remains largely unexplored in WWTPs. Our custom pipeline for detecting nosZ provides near-full-length genes with detailed information on secretory pathways and accessory nos genes. Using these genes as templates, we developed taxonomically diverse clade-specific primers that generate nosZ amplicons for phylogenetic annotation and gene-to-MAG linkage. This approach improves detection and expands the discovery of novel sequences, highlighting the prevalence of non-denitrifying N2O reducers and their potential as N2O sinks. These findings have the potential to optimize nitrogen removal processes and mitigate greenhouse gas emissions from WWTPs by fully harnessing the capabilities of the microbial communities.

Proteomic Changes in Methicillin-Resistant Staphylococcus aureus Exposed to Cannabinoids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to ß-lactam antibiotics complicates treatment due to the limited number of antibiotics with activity against MRSA. To investigate development of alternative therapeutics, the mechanisms that mediate antibiotic resistance in MRSA need to be fully understood. In this study, MRSA cells were subjected to antibiotic stress from methicillin in combination with three cannabinoid compounds and analyzed using proteomics to assess the changes in physiology. Subjecting MRSA to nonlethal levels of methicillin resulted in an increased production of penicillin-binding protein 2 (PBP2). Exposure to cannabinoids showed antibiotic activity against MRSA, and differential proteomics revealed reduced levels of proteins involved in the energy production as well as PBP2 when used in combination with methicillin.

Impact of water quality parameters on geosmin levels and geosmin producers in European recirculating aquaculture systems.

AIMS: Geosmin is associated with off-flavour problems in recirculating aquaculture systems (RAS) and represents an economic problem for the aquaculture industry. This study aims at investigating factors influencing the composition of the bacterial microbiota, in particular the presence of geosmin producers and the environmental and farming factors favouring geosmin accumulation. METHODS AND RESULTS: Several water quality parameters were correlated to the composition of the microbiota with special emphasis on the presence of geosmin producers within 26 different RAS from four European countries. Three novel groups of geosmin-producing bacteria were quantified to identify potential correlations with geosmin concentration. CONCLUSIONS: The microbiome differed significantly between systems. However, phosphate levels, calcium levels and redox potential correlated to geosmin concentration in the water and the presence of the Actinomycetales geosmin-producers but not with the presence of other groups of geosmin-producing bacteria. Oxygen levels and conductivity were found to negatively correlate with geosmin concentration. A large proportion of the detected geosmin producers represented novel taxonomic groups not previously linked with this activity. SIGNIFICANCE AND IMPACT OF THE STUDY: These results improve our understanding of the diversity of microbiota in RAS and the water quality parameters favouring the populations of geosmin-producing bacteria and the production of geosmin.

A cross sectional study on airborne inhalable microorganisms, endotoxin, and particles in pigeon coops - Risk assessment of exposure.

Pigeon breeding is associated with symptoms of the airways. The aim of this study is to illuminate the bacteriological and toxicological characteristics of airborne dust in pigeon coops. Airborne dust was sampled in 31 urban pigeon coops with homing and fancy pigeons, and following the dust was characterized. In total 141 different bacterial species were identified using MALDI-TOF MS, and of these 11 species are classified in risk group 2. Of the cultivable bacteria, Staphylococcus equorum was present in the highest concentration. Microorganisms in the dust were able to form biofilm, and the amount correlated positively with the number of bacteria. Next generation sequencing showed 180 genera with Acinetobacter in highest reads. On average 999 ± 225 ZOTUs were observed per sample with a Shannon-Wiener biodiversity index of 6.17 ± 0.24. Of the identified species the following have previously been suggested as causative agents of extrinsic allergic alveolitis: Alcaligenes faecalis, Bacillus subtilis, Pantoea agglomerans, Sphingobacterium spiritivorum, Thermoactinomyces sp., and Streptomyces albus. Staphylococcus was present on particles with sizes between 1.1 and > 7.0 µm with a geometric mean diameter of particles on 4.7 ± 1.1 µm. Concentrations of airborne endotoxin and dust were elevated compared to references, and the geometric mean concentrations were 102 EU/m3 and 1.07 mg dust/m3, respectively. Upon exposure to the airborne dust human granulocytes produced Reactive Oxidative Species during the first 5 min, and then no further reaction was observed. The concentrations of bacteria in general, Staphylococcus spp., and endotoxin and biodiversity were associated significantly with season, temperature and/or relative humidity, but not with type or density of pigeons. The bacterial composition and biodiversity indices were not affected by type of pigeon. In conclusion, the exposure to bacteria and endotoxin in pigeon houses should not be neglected in the evaluation of causative agents of airways symptoms among pigeon breeders.

Physiological Responses of Aspergillus niger Challenged with Itraconazole.

Aspergillus niger is an opportunistic pathogen commonly found in a variety of indoor and outdoor environments. An environmental isolate of A. niger from a pig farm was resistant to itraconazole, and in-depth investigations were conducted to better understand cellular responses that occur during growth when this pathogen is exposed to an antifungal. Using a combination of cultivation techniques, antibiotic stress testing, and label-free proteomics, this study investigated the physiological and metabolic responses of A. niger to sublethal levels of antifungal stress. Challenging A. niger with itraconazole inhibited growth, and the MIC was estimated to be > 16 mg · liter-1 Through the proteome analysis, 1,305 unique proteins were identified. During growth with 2 and 8 mg · liter-1 itraconazole, a total of 91 and 50 proteins, respectively, were significantly differentially expressed. When challenged with itraconazole, A. niger exhibited decreased expression of peroxidative enzymes, increased expression of an ATP-binding cassette (ABC) transporter most likely involved as an azole efflux pump, and inhibited ergosterol synthesis; however, several ergosterol biosynthesis proteins increased in abundance. Furthermore, reduced expression of proteins involved in the production of ATP and reducing power from both the tricarboxylic acid (TCA) and glyoxylate cycles was observed. The mode of action of triazoles in A. niger therefore appears more complex than previously anticipated, and these observations may help highlight future targets for antifungal treatment.

A cohort study of cucumber greenhouse workers' exposure to microorganisms as measured using NGS and MALDI-TOF MS and biomarkers of systemic inflammation.

Work in greenhouses entails exposure to airborne fungi and bacteria. The aims of this study are to obtain knowledge about whether exposure to fungal and bacterial genera and species during work in a cucumber greenhouse is affected by work tasks, and whether a cohort of greenhouse workers' serum levels of serum amyloid A (SAA) and C-reactive protein (CRP), biomarkers of systemic inflammation, are associated with this. Data on personal exposure to airborne fungal and bacterial species measured over 4 years as well as serum levels of SAA and CRP sampled over two years were analyzed. For data analysis, the main work tasks were grouped into three different groups, called 'grouped work task'. Microorganisms were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) and next-generation sequencing (NGS). The 'daily exposure' of greenhouse workers' were as follows: 4.8 × 104 CFU bacteria/m3, 1.4 × 106 CFU fungi/m3, and 392 EU/m3 of endotoxin. Workers were exposed to many different microbial species including several species within the genera Acinetobacter, Bacillus, Microbacterium, Pseudomonas, Staphylococcus, and Streptomyces. The genera Ralstonia and Cladosporium were found in most samples. The exposure levels as well as the microbial composition were associated significantly with grouped work task and season with high exposures during tasks in close contact with mature and old plants and in the autumn. CRP and SAA levels were also associated with exposure level and grouped work tasks. The Shannon-Wiener indices were not different in the 3 'grouped work tasks'. Several specific species including e.g. Halomonas elongata, Stenotrophomonas maltophilia, Podosphaera fusca, and Wallemia spp. were found frequently or in high concentrations in the exposures associated with the highest levels of CRP and SAA. The microorganisms S. maltophilia, P. fusca, and Wallemia spp. were also found on the cucumber plant leaves. In conclusion, both exposure level and the species composition seem to have an effect on the serum levels of CRP and SAA of exposed workers. The greenhouse workers were exposed to only a few species characterized as human pathogens.

Integrated genome-wide investigations of the housefly, a global vector of diseases reveal unique dispersal patterns and bacterial communities across farms.

BACKGROUND: Houseflies (Musca domestica L.) live in intimate association with numerous microorganisms and is a vector of human pathogens. In temperate areas, houseflies will overwinter in environments constructed by humans and recolonize surrounding areas in early summer. However, the dispersal patterns and associated bacteria across season and location are unclear. We used genotyping-by-sequencing (GBS) for the simultaneous identification and genotyping of thousands of Single Nucleotide Polymorphisms (SNPs) to establish dispersal patterns of houseflies across farms. Secondly, we used 16S rRNA gene amplicon sequencing to establish the variation and association between bacterial communities and the housefly across farms. RESULTS: Using GBS we identified 18,000 SNPs across 400 individuals sampled within and between 11 dairy farms in Denmark. There was evidence for sub-structuring of Danish housefly populations and with genetic structure that differed across season and sex. Further, there was a strong isolation by distance (IBD) effect, but with large variation suggesting that other hidden geographic barriers are important. Large individual variations were observed in the community structure of the microbiome and it was found to be dependent on location, sex, and collection time. Furthermore, the relative prevalence of putative pathogens was highly dependent on location and collection time. CONCLUSION: We were able to identify SNPs for the determination of the spatiotemporal housefly genetic structure, and to establish the variation and association between bacterial communities and the housefly across farms using novel next-generation sequencing (NGS) techniques. These results are important for disease prevention given the fine-scale population structure and IBD for the housefly, and that individual houseflies carry location specific bacteria including putative pathogens.

Occurrence of Cyanobacteria and microcystins in hydroelectric reservoirs used for fish farming.

Fish farming can have a negative impact on water quality and aquatic organisms due to emerging blooms of Cyanobacteria and the production of cyanotoxins. In this study, the effect of aquaculture in hydroelectric reservoirs in Brazil was evaluated in six fish farms and in upstream and downstream water through analysis of the microbiome, Cyanobacteria and microcystin concentrations. Synechococcus and Microcystis were observed at all six locations, while Limnothrix was also observed abundantly at two locations. An increase in the relative abundance of Cyanobacteria inside the fish farms was observed at two locations, while an increase of Cyanobacteria was observed in downstream at five of the six locations. Microcystins were detected in significant and high values in all locations, with concentrations up to 1.59 µg/L. The trend in microcystin concentrations was mirrored in copy numbers of the mcyE gene (encodes microcystin synthetase) and presence of Microcystis, but not in any of the other observed cyanobacterial groups. In summary, the study shows that aquaculture production influenced the water microbiome inside and downstream the fish farms, and a direct correlation was found between mcyE gene copies, microcystin production and abundance of Microcystis, but not for the total abundance of Cyanobacteria.

Microbial species and biodiversity in settling dust within and between pig farms.

The airborne fungal and bacterial species present in pig farm dust have not been well characterised even though these bioaerosols are known to cause inflammation and other airway maladies. In this study, the microbial species and composition in airborne dust within and between pig farms were investigated. Passively sedimenting dust from six pig farms were collected using electrostatic dust collectors. The bacterial and fungal species were identified using matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and next generation sequencing (NGS). Dust samples taken within the same stable section revealed high resemblance and stability. Constrained statistical analysis of the microbial community compositions indicated that the types of stable did not appear to have a great effect on the bacterial and fungal ß-diversity. In contrast to this, the farm from which samples were taken appeared to have the greatest effect on the bacterial ß-diversity, but this trend was not observed for the fungal ß-diversity. The most common bacteria and fungi according to NGS data were anaerobes typically associated with the pig intestinal tract and yeasts respectively. Bacterial sedimentation varied at a rate between 103 and 109 CFU/m2/day, with the most common species after aerobic incubation being Aerococcus viridans and Staphylococcus equorum, while Clostridium perfringens and Staphylococcus simulans were the most common species after anaerobic incubation. A total of 28 different species of bacteria and fungi were classifiable as pathogens. In conclusion, the biodiversity in pig farm dust shows a high diversity of bacterial species. However, samples from the same stable section resembled each other, but also different sections within the same farm also resembled each other, thus indicating a high degree of community stability in the dust source. In regards to fungal identification, the biodiversity was observed to be similar between samples from different stable sections and farms, indicating a higher degree of similarities in the mycobiomes found across pig farms studied.

Enhancing metaproteomics--The value of models and defined environmental microbial systems.

Metaproteomics--the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time--has provided new features to study complex microbial communities in order to unravel these "black boxes." New technical challenges arose that were not an issue for classical proteome analytics before that could be tackled by the application of different model systems. Here, we review different current and future model systems for metaproteome analysis. Following a short introduction to microbial communities and metaproteomics, we introduce model systems for clinical and biotechnological research questions including acid mine drainage, anaerobic digesters, and activated sludge. Model systems are useful to evaluate the challenges encountered within (but not limited to) metaproteomics, including species complexity and coverage, biomass availability, or reliable protein extraction. The implementation of model systems can be considered as a step forward to better understand microbial community responses and ecological functions of single member organisms. In the future, improvements are necessary to fully explore complex environmental systems by metaproteomics.

Identification of active denitrifiers in full-scale nutrient removal wastewater treatment systems.

Denitrification is essential to the removal of nitrogen from wastewater during treatment, yet an understanding of the diversity of the active denitrifying bacteria responsible in full-scale wastewater treatment plants (WWTPs) is lacking. In this study, stable-isotope probing (SIP) was applied in combination with microautoradiography (MAR)-fluorescence in situ hybridization (FISH) to identify previously unrecognized active denitrifying phylotypes in a full-scale WWTP with biological N and P removal. Acknowledging that different denitrifiers will have specific carbon source preferences, a fully (13)C-labelled complex substrate was used for SIP incubations, under nitrite-reducing conditions, in order to maximize the capture of the potentially metabolically diverse denitrifiers likely present. Members of the Rhodoferax, Dechloromonas, Sulfuritalea, Haliangium and Thermomonas were represented in the 16S rRNA gene clone libraries from DNA enriched in (13)C, with FISH probes optimized here for their in situ characterization. FISH and MAR confirmed that they were all active denitrifiers in the community. The combined approach of SIP and MAR-FISH represents an excellent approach for identifying and characterizing an un-described diversity of active denitrifiers in full-scale systems.

In vivo gene expression in a Staphylococcus aureus prosthetic joint infection characterized by RNA sequencing and metabolomics: a pilot study.

BACKGROUND: Staphylococcus aureus gene expression has been sparsely studied in deep-sited infections in humans. Here, we characterized the staphylococcal transcriptome in vivo and the joint fluid metabolome in a prosthetic joint infection with an acute presentation using deep RNA sequencing and nuclear magnetic resonance spectroscopy, respectively. We compared our findings with the genome, transcriptome and metabolome of the S. aureus joint fluid isolate grown in vitro. RESULT: From the transcriptome analysis we found increased expression of siderophore synthesis genes and multiple known virulence genes. The regulatory pattern of catabolic pathway genes indicated that the bacterial infection was sustained on amino acids, glycans and nucleosides. Upregulation of fermentation genes and the presence of ethanol in joint fluid indicated severe oxygen limitation in vivo. CONCLUSION: This single case study highlights the capacity of combined transcriptome and metabolome analyses for elucidating the pathogenesis of prosthetic infections of major clinical importance.

Proteomic data reveal a physiological basis for costs and benefits associated with thermal acclimation.

Physiological adaptation through acclimation is one way to cope with temperature changes. Biochemical studies on acclimation responses in ectotherms have so far mainly investigated consequences of short-term acclimation at the adult stage and focussed on adaptive responses. Here, we assessed the consequences of rearing Drosophila melanogasterat low (12°C), benign (25°C) and high (31°C) temperatures. We assessed cold and heat tolerance and obtained detailed proteomic profiles of flies from the three temperatures. The proteomic profiles provided a holistic understanding of the underlying biology associated with both adaptive and non-adaptive temperature responses. Results show strong benefits and costs across tolerances: rearing at low temperature increased adult cold tolerance and decreased adult heat tolerance and vice versa with development at high temperatures. In the proteomic analysis, we were able to identify and quantify a large number of proteins compared with previous studies on ectotherms (1440 proteins across all replicates and rearing regimes), enabling us to extend the proteomic approach using enrichment analyses. This gave us detailed information on individual proteins, as well as pathways affected by rearing temperature, pinpointing potential mechanisms responsible for the strong costs and benefits of rearing temperature on functional phenotypes. Several well-known heat shock proteins, as well as proteins not previously associated with thermal stress, were among the differentially expressed proteins. Upregulation of proteasome proteins was found to be an important adaptive process at high-stress rearing temperatures, and occurs at the expense of downregulation of basal metabolic functions.

Microbial diversity in bioaerosol samples causing ODTS compared to reference bioaerosol samples as measured using Illumina sequencing and MALDI-TOF.

The importance of the microbial diversity of bioaerosols in relation to occupational exposure and work related health symptoms is not known. The aim of this paper is to gain knowledge on the bacterial and fungal communities in dust causing organic dust toxic syndrome (ODTS) and in reference dust not causing ODTS. Bacterial and fungal communities were described in personal exposure samples from grass seed workers developing ODTS, in dust generated from grass seeds causing ODTS and in dust generated from reference seeds not causing ODTS. Amplicon sequencing of the bacterial 16S rRNA gene and the fungal ITS region, as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) were used for identification of fungi and bacteria in personal exposure samples and in dust samples from grass seeds causing ODTS and in dust from reference grass seeds. Furthermore, activities of enzymes were measured in the same samples. The sequencing data revealed more than 150 bacterial and 25 fungal genera present in each sample. Streptomyces spp., Aspergillus fumigatus and Rhizopus microsporus were dominating in the dust causing ODTS but not in the reference dust. The dustiness in terms of Mucor sp. and R. microsporus were 100-1000 times higher for problematic seeds compared to reference seeds. The bacterial species in the dust causing ODTS included pathogenic species such as Klebsiella pneumonia and Streptomyces pneumonia, and it contained increased concentrations of total protein, serine protease, chitinase, and ß-glucosidase. Twenty-three bacterial genera covered more than 50% of the total reads in the personal and problematic seed dust. These 23 genera accounted for less than 7% of the total reads in the reference seed dust. The microbial community of the dust from the problematic seeds showed great similarities to that from the personal air samples from the workers. In conclusion, we have shown for the first time a shift in the microbial community in aerosol samples that caused ODTS compared to the reference samples that did not cause the ODTS. Furthermore, elevated enzyme activities were found in the dust causing ODTS.

Bioremediation strategies for removal of residual atrazine in the boreal groundwater zone.

Strategies for bioremediation of atrazine, a pesticide commonly polluting groundwater in low concentrations, were studied in two boreal nonagricultural soils. Atrazine was not mineralized in soil without bioremediation treatments. In biostimulation treatment with molasses, up to 52% of atrazine was mineralized at 10 °C, even though the degradation gene copy numbers did not increase. Incubations with radioactively labeled atrazine followed by microautoradiographic analysis revealed that bioremediation strategies increased the relative proportion of active degraders from 0.3 up to 1.9% of the total bacterial count. These results indicate that atrazine degradation might not solely be facilitated by atzA/trzN-atzB genes. In combined biostimulation treatment using citrate or molasses and augmentation with Pseudomonas citronellolis ADP or Arthrobacter aurescens strain TC1, up to 76% of atrazine was mineralized at 30 °C, and the atrazine degradation gene numbers increased up to 10(7) copies g(-1) soil. Clone libraries from passive samplers in groundwater monitoring wells revealed the presence of phylogenetic groups formerly shown to include atrazine degraders, and the presence of atrazine degradation genes atzA and atzB. These results show that the mineralization of low concentrations of atrazine in the groundwater zone at low temperatures is possible by bioremediation treatments.

Degradation of PPCPs in activated sludge from different WWTPs in Denmark.

Pharmaceuticals and Personal care products (PPCPs) are often found in effluents from wastewater treatment plants (WWTPs) due to insufficient removal during wastewater treatment processes. To understand the factors affecting the removal of PPCPs in classical activated sludge WWTPs, the present study was performed to assess the removal of frequently occurring pharmaceuticals (Naproxen, Fenoprofen, Ketoprofen, Dichlofenac, Carbamazepine) and the biocide Triclosan in activated sludge from four different Danish WWTPs. The respective degradation constants were compared to operational parameters previous shown to be of importance for degradation of micropollutants such as biomass concentration, and sludge retention time (SRT). The most rapid degradation, was observed for NSAID pharmaceuticals (55-90% for Fenoprofen, 77-94% for Ketoprofen and 46-90% for Naproxen), followed by Triclosan (61-91%), while Dichlofenac and Carbamazepine were found to be persistent in the systems. Degradation rate constants were calculated as 0.0026-0.0407 for NSAID pharmaceuticals and 0.0022-0.0065 for triclosan. No relationships were observed between degradation rates and biomass concentrations in the diverse sludges. However, for the investigated PPCPs, the optimal SRT was within 14-20 days (for these values degradation of these PPCPs was the most efficient). Though all of these parameters influence the degradation rate, none of them seems to be overall decisive. These observations indicate that the biological composition of the sludge is more important than the design parameters of the respective treatment plant.

Quantifying contribution of synthrophic acetate oxidation to methane production in thermophilic anaerobic reactors by membrane inlet mass spectrometry.

A unique method was developed and applied for monitoring methanogenesis pathways based on isotope labeled substrates combined with online membrane inlet quadrupole mass spectrometry (MIMS). In our study, a fermentation sample from a full-scale biogas plant fed with pig and cattle manure, maize silage, and deep litter was incubated with 100 mM of [2-(13)C] sodium acetate under thermophilic anaerobic conditions. MIMS was used to measure the isotopic distribution of dissolved CO2 and CH4 during the degradation of acetate, while excluding interference from water by applying a cold trap. After 6 days of incubation, the proportion of methane derived from reduction of CO2 had increased significantly and reached up to 87% of total methane, suggesting that synthrophic acetate oxidation coupled to hydrogenotrophic methanogenesis (SAO-HM) played an important role in the degradation of acetate. This study provided a new approach for online quantification of the relative contribution of methanogenesis pathways to methane production with a time resolution shorter than one minute. The observed contribution of SAO-HM to methane production under the tested conditions challenges the current widely accepted anaerobic digestion model (ADM1), which strongly emphasizes the importance of the acetoclastic methanogenesis.

Complete pipeline for Oxford Nanopore Technology amplicon sequencing (ONT-AmpSeq): from pre-processing to creating an operational taxonomic unit table.

Amplicon sequencing has long served as a robust method for characterising microbial communities, and despite inherent resolution limitations, it remains a preferred technique, offering cost- and time-effective insights into bacterial compositions. Here, we introduce ONT-AmpSeq, a user-friendly pipeline designed for processing amplicon sequencing data generated from Oxford Nanopore Technology (ONT) devices. Our pipeline enables efficient creation of taxonomically annotated operational taxonomic unit (OTU) tables from ONT sequencing data, with the flexibility to multiplex amplicons on the same barcode. The pipeline encompasses six main steps-statistics, quality filtering, alignment, clustering, polishing, and taxonomic classification-integrating various state-of-the-art software tools. We provide a detailed description of each step, along with performance tests and robustness evaluations using both test data and a ZymoBIOMICS® Microbial Community Standard mock community dataset. Our results demonstrate the ability of ONT-AmpSeq to effectively process ONT amplicon data, offering valuable insights into microbial community composition. Additionally, we discuss the influence of polishing tools on taxonomic insight and the impact of taxonomic annotation methods on the derived microbial composition. Overall, ONT-AmpSeq represents a comprehensive solution for analysing ONT amplicon sequencing data, facilitating streamlined and reliable microbial community analysis. The pipeline, along with test data, is freely available for public use.

A Review of the Occurrence of Metals and Xenobiotics in European Hedgehogs (Erinaceus europaeus).

Monitoring data from several European countries indicate that European hedgehog (Erinaceus europaeus) populations are declining, and research exploring the causes of the decline, including exposure to potentially harmful xenobiotics and metals, may inform conservation initiatives to protect this species in the wild. Hedgehogs are ground-dwelling mammals, feeding on a range of insects, slugs, snails, and earthworms, as well as eggs, live vertebrates, and carrion, including carcasses of apex predator species representing higher levels of the food chain. Consequently, hedgehogs come into close contact with contaminants present in their habitats and prey. This review investigated the studies available on the subject of the occurrence of metals and organic xenobiotics in hedgehogs. This study found that a vast range of different pesticides; persistent organic pollutants (POPs), including organochlorine compounds and brominated flame retardants (BFRs); as well as toxic heavy metals could be detected. Some compounds occurred in lethal concentrations, and some were associated with a potential adverse effect on hedgehog health and survival. Due to their ecology, combined with the opportunity to apply non-invasive sampling techniques using spines as sampling material, we suggest that the European hedgehog is a relevant bioindicator species for monitoring the exposure of terrestrial wildlife to potential toxicants in urban and rural environments.

Eat seldom is better than eat frequently: Pharmaceuticals degradation kinetics, enantiomeric profiling and microorganisms in moving bed biofilm reactors are affected by feast famine cycle times.

Feast-famine (FF) regimes improved the removal of recalcitrant pharmaceuticals in moving bed biofilm reactors (MBBRs), but the optimal FF cycle remained unresolved. The effects of FF cycle time on the removal of bulk substrates (organic carbon and nitrogen) and trace pharmaceuticals by MBBR are systematically evaluated in this study. The feast to famine ratio was fixed to 1:2 to keep the same loading rate, but the time for the FF cycles varied from 18 h to 288 h. The MBBR adapted to the longest FF cycle time (288 h equaling 48 × HRT) resulted in significantly higher degradation rates (up to +183%) for 12 out of 28 pharmaceuticals than a continuously fed (non-FF) reactor. However, other FF cycle times (18, 36, 72 and 144 h) only showed a significant up-regulation for 2-3 pharmaceuticals compared to the non-FF reactor. Enantioselective degradation of metoprolol and propranolol occurred in the second phase of a two phase degradation, which was different for the longer FF cycle time. N-oxidation and N-demethylation pathways of tramadol and venlafaxine differed across the FF cycle time suggestin the FF cycle time varied the predominant transformation pathways of pharmaceuticals. The abundance of bacteria in the biofilms varied considerably between different FF cycle times, which possibly caused the biofilm to remove more recalcitrant bulk organic C and pharmaceuticals under long cycle times.