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Biological reduction of CO 2 into CH 4 by exogenous addition of H 2 is a promising technology for upgrading biogas into higher CH 4 content. The aim of this work was to study the feasibility of exogenous H 2 addition for an in situ biogas upgrading through biological conversion of the biogas CO 2 into CH 4. Moreover, this study employed systematic study with isotope analysis for providing comprehensive evidence on the underlying pathways of CH 4 production and upstream processes. Batch reactors were inoculated with digestate originating from a full-scale biogas plant and fed once with maize leaf substrate. Periodic addition of H 2 into the headspace resulted in a completely consumption of CO 2 and a concomitant increase in CH 4 content up to 89%. The microbial community and isotope analysis shows an enrichment of hydrogenotrophic Methanobacterium and the key role of hydrogenotrophic methanogenesis for biogas upgrading to higher CH 4 content. Excess H 2 was also supplied to evaluate its effect on overall process performance. The results show that excess H 2 addition resulted in accumulation of H 2 , depletion of CO 2 and inhibition of the degradation of acetate and other volatile fatty acids (VFA). A systematic isotope analysis revealed that excess H 2 supply led to an increase in dissolved H 2 to the level that thermodynamically inhibit the degradation of VFA and stimulate homo-acetogens for production of acetate from CO 2 and H 2 . The inhibition was a temporary effect and acetate degradation resumed when the excess H 2 was removed as well as in the presence of stoichiometric amount of H 2 and CO 2 . This inhibition mechanism underlines the importance of carefully regulating the H 2 addition rate and gas retention time to the CO 2 production rate, H 2 -uptake rate and growth of hydrogenotrophic methanogens in order to achieve higher CH 4 content without the accumulation of acetate and other VFA. [ABSTRACT FROM AUTHOR]

Earthy off-flavour, caused by geosmin, constitutes an economic problem in recirculating aquaculture systems (RAS). Cyanobacteria , Streptomyces and myxobacteria are thought to be the main geosmin-producing bacteria in aquatic systems. In this study, the phylogeny of the gene involved in the geosmin production, geosmin synthase gene ( geoA ), was investigated. The results revealed an absence of multiple horizontal gene transfer, and therefore, the gene might be suited as a molecular marker to investigate the diversity of putatively geosmin-producing bacteria. Degenerate primers targeting the major phylogenetic groups of geoA were used to describe the geosmin-producing populations in water and biofilm. Gene libraries from six RAS, collected in Scotland and Denmark, were investigated. This revealed at least two novel, deep-branching lineages. The most dominant geoA -containing bacteria present in the investigated RAS affiliated with myxobacteria, Sorangium and Actinobacteria . TaqMan qPCR approaches were designed to quantify four novel groups of geosmin-producing bacteria. The results showed that geosmin-producing bacteria are present in the biofilters and water phase, and although they constitute only 0.007%–0.9% of the total bacteria, they cause significant off-flavour problems. This study provides an insight into the phylogeny and quantities of geosmin-producing microbiota in full-scale recirculated aquaculture systems. [ABSTRACT FROM AUTHOR]

Surplus electricity from fluctuating renewable power sources may be converted to CH 4 via biomethanisation in anaerobic digesters. The reactor performance and response of methanogen population of mixed-culture reactors was assessed during pulsed H 2 injections. Initial H 2 uptake rates increased immediately and linearly during consecutive pulse H 2 injections for all tested injection rates (0.3 to 1.7 L H2 /L sludge /d), while novel high throughput mcrA sequencing revealed an increased abundance of specific hydrogenotrophic methanogens. These findings illustrate the adaptability of the methanogen population to H 2 injections and positively affects the implementation of biomethanisation. Acetate accumulated by a 10-fold following injections exceeding a 4:1 H 2 :CO 2 ratio and may act as temporary storage prior to biomethanisation. Daily methane production decreased for headspace CO 2 concentrations below 12% and may indicate a high sensitivity of hydrogenotrophic methanogens to CO 2 limitation. This may ultimately decide the biogas upgrading potential which can be achieved by biomethanisation. [ABSTRACT FROM AUTHOR]

The evolution and population dynamics of complex anaerobic microbial communities in anaerobic digesters were investigated during stable operation and recovery after prolonged starvation. Three thermophilic reactor systems fed with cattle manure were operated continuously in parallel for 167 days. Significant changes in the microbial communities were observed for both the bacterial and archaeal populations as the reactor systems were subjected to changing feeding regimes. The ecosystems developed from being relatively similar in structure to more specialised communities, with large population shifts within the acetogenic and methanogenic communities, which appeared to shift towards the hydrogenotrophic methanogenesis pathway. All reactor systems showed signs of adaptation to a harsher environment under high VFA, H 2 S and ammonia concentrations, but remained at a lower degree of stability after 45 days of recovery compared to stable period of operation before starvation. [ABSTRACT FROM AUTHOR]

• Ensifer adhaerens and a Chelatococcus sp. enriched using acesulfame as C-source. • Enzymatically catalyzed degradation of the artificial sweetener in lysates. • Protein fractionation via two-dimensional fast protein liquid chromatography. • Protein expression profiling of acesulfame degraders via shotgun proteomics. • Identification of enzyme candidates likely involved in the transformation pathway. The removal of organic micropollutants in municipal wastewater treatment is an extensively studied field of research, but the underlying enzymatic processes have only been elucidated to a small extent so far. In order to shed more light on the enzymatic degradation of the artificial sweetener acesulfame (ACE) in this context, we enriched two bacterial taxa which were not yet described to be involved in the degradation of ACE, an unknown Chelatococcus species and Ensifer adhaerens , by incubating activated sludge in chemically defined media containing ACE as sole carbon source. Cell-free lysates were extracted, spiked with ACE and analyzed via target LC-MS/MS, demonstrating for the first time enzymatically catalyzed ACE degradation outside of living cells. Fractionation of the lysate via two-dimensional fast protein liquid chromatography (FPLC) succeeded in a partial separation of the enzymes catalyzing the initial transformation reaction of ACE from those catalyzing the further transformation pathway. Thereby, an accumulation of the intermediate transformation product acetoacetamide-n-sulfonic acid (ANSA) in the ACE-degrading fractions was achieved, providing first quantitative evidence that the cleavage of the sulfuric ester moiety of ACE is the initial transformation step. The metaproteome of the enrichments was analyzed in the FPLC fractions and in the unfractionated lysate, using shotgun proteomics via UHPLC-HRMS/MS and label-free quantification. The comparison of protein abundances in the FPLC fractions to the corresponding ACE degradation rates revealed a metallo-β-lactamase fold metallo-hydrolase as most probable candidate for the enzyme catalyzing the initial transformation from ACE to ANSA. This enzyme was by far the most abundant of all detected proteins and amounted to a relative protein abundance of 91% in the most active fraction after the second fractionation step. Moreover, the analysis of the unfractionated lysate resulted in a list of further proteins possibly involved in the transformation of ACE, most striking a highly abundant amidase likely catalyzing the further transformation of ANSA, and an ABC transporter substrate-binding protein that may be involved in the uptake of ACE into the cell. [Display omitted] [ABSTRACT FROM AUTHOR]

Organic micropollutants (OMPs) such as pharmaceuticals are persistent pollutants that are only partially degraded in waste water treatment plants (WWTPs). In this study, a membrane bioreactor (MBR) system was used as a polishing step on a full-scale WWTP, and its ability to remove micropollutants was examined together with the development and stability of the microbial community. Two stages of operation were studied during a period of 9 months, one with (S1) and one without (S2) the addition of exogenous OMPs. Ibuprofen and naproxen had the highest degradation rates with values of 248 μg/g VSS ·h and 71 μg/g VSS ·h, whereas diclofenac was a more persistent OMP (7.28 μg/g VSS ·h). Mineralization of 14 C-labeled OMPs in batch kinetic experiments indicates that higher removal rates (∼0.8 ng/mg T SS ·h) with a short lag phase can be obtained when artificial addition of organic micropollutants was performed. Similar microbial populations dominated S1 and S2, despite the independent operations. Hydrogenophaga , Nitrospira , p55-a5, the actinobacterial Tetrasphaera , Propionicimonas , Fodinicola , and Candidatus Microthrix were the most abundant groups in the polishing MBR. Finally, potential microbial candidates for ibuprofen and naproxen degradation are proposed. [ABSTRACT FROM AUTHOR]

The implementation of autotrophic anaerobic ammonium oxidation processes for the removal of nitrogen from municipal wastewater (known as “mainstream anammox”) bears the potential to bring wastewater treatment plants close to energy autarky. The aim of the present work was to assess the long-term stability of partial nitritation/anammox (PN/A) processes operating at low temperatures and their reliability in meeting nitrogen concentrations in the range of typical discharge limits below 2 mg NH 4 - N · L − 1 and 10 mg Ntot ·L −1 . Two main 12-L sequencing batch reactors were operated in parallel for PN/A on aerobically pre-treated municipal wastewater (21 ± 5 mg NH 4 - N · L − 1 and residual 69 ± 19 mg CODtot ·L −1 ) for more than one year, including over 5 months at 15 °C. The two systems consisted of a moving bed biofilm reactor (MBBR) and a hybrid MBBR (H-MBBR) with flocculent biomass. Operation at limiting oxygen concentrations (0.15–0.18 mg O 2 · L − 1 ) allowed stable suppression of the activity of nitrite-oxidizing bacteria at 15 °C with a production of nitrate over ammonium consumed as low as 16% in the MBBR. Promising nitrogen removal rates of 20–40 mg N ·L −1 ·d −1 were maintained at hydraulic retention times of 14 h. Stable ammonium and total nitrogen removal efficiencies over 90% and 70% respectively were achieved. Both reactors reached average concentrations of total nitrogen below 10 mg N ·L −1 in their effluents, even down to 6 mg N ·L −1 for the MBBR, with an ammonium concentration of 2 mg N ·L −1 (set as operational threshold to stop aeration). Furthermore, the two PN/A systems performed almost identically with respect to the biological removal of organic micropollutants and, importantly, to a similar extent as conventional treatments. A sudden temperature drop to 11 °C resulted in significant suppression of anammox activity, although this was rapidly recovered after the temperature was increased back to 15 °C. Analyses of 16S rRNA gene-targeted amplicon sequencing revealed that the anammox guild of the bacterial communities of the two systems was composed of the genus “ Candidatus Brocadia”. The potential of PN/A systems to compete with conventional treatments for biological nutrients removal both in terms of removal rates and overall effluent quality was proven. [ABSTRACT FROM AUTHOR]

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 R hodoferax, D echloromonas, S ulfuritalea, H aliangium and T hermomonas were represented in the 16 S 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. [ABSTRACT FROM AUTHOR]

Direct treatment of municipal wastewater (MWW) based on anaerobic ammonium oxidizing (anammox) bacteria holds promise to turn the energy balance of wastewater treatment neutral or even positive. Currently, anammox processes are successfully implemented at full scale for the treatment of high-strength wastewaters, whereas the possibility of their mainstream application still needs to be confirmed. In this study, the growth of anammox organisms on aerobically pre-treated municipal wastewater (MWW pre-treated ), amended with nitrite, was proven in three parallel reactors. The reactors were operated at total N concentrations in the range 5–20 mg N ∙L −1 , as expected for MWW. Anammox activities up to 465 mg N ∙L −1 ∙d −1 were reached at 29 °C, with minimum doubling times of 18 d. Lowering the temperature to 12.5 °C resulted in a marked decrease in activity to 46 mg N ∙L −1 ∙d −1 (79 days doubling time), still in a reasonable range for autotrophic nitrogen removal from MWW. During the experiment, the biomass evolved from a suspended growth inoculum to a hybrid system with suspended flocs and wall-attached biofilm. At the same time, MWW pre-treated had a direct impact on process performance. Changing the influent from synthetic medium to MWW pre-treated resulted in a two-month delay in net anammox growth and a two to three-fold increase in the estimated doubling times of the anammox organisms. Interestingly, anammox remained the primary nitrogen consumption route, and high-throughput 16S rRNA gene-targeted amplicon sequencing analyses revealed that the shift in performance was not associated with a shift in dominant anammox bacteria (“ Candidatus Brocadia fulgida”). Furthermore, only limited heterotrophic denitrification was observed in the presence of easily biodegradable organics (acetate, glucose). The observed delays in net anammox growth were thus ascribed to the acclimatization of the initial anammox population or/and the development of a side population beneficial for them. Additionally, by combining microautoradiography and fluorescence in situ hybridization it was confirmed that the anammox organisms involved in the process did not directly incorporate or store the amended acetate and glucose. In conclusion, these investigations strongly support the feasibility of MWW treatment via anammox. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

Successful application of bioaugmentation for enhanced degradation of environmental pollutants is often limited by the lack of methods to monitor the survival and activity of individual bioaugmentation strains. However, recent advancements in sequencing technologies and molecular techniques now allow us to address these limitations. Here a complementing set of general applicable molecular methods are presented that provides detailed information on the performance of individual bioaugmentation strains under in situ conditions. The approach involves genome sequencing to establish highly specific qPCR and RT-qPCR tools for cell enumerations and expression of involved genes, stable isotope probing to follow growth on the target compounds and GFP-tagging to visualize the bioaugmentation strains directly in samples, all in combination with removal studies of the target compounds. The concept of the approach is demonstrated through a case study involving degradation of aromatic hydrocarbons in activated sludge augmented with the bioaugmentation strain Pseudomonas monteilii SB3078. [ABSTRACT FROM AUTHOR]

Enhanced biological phosphorus removal (EBPR) is one of the most advanced and complicated wastewater treatment processes applied today, and it is becoming increasingly popular worldwide as a sustainable way to remove and potentially reuse P. It is carried out by complex microbial communities consisting primarily of uncultured microorganisms. The EBPR process is a well-studied system with clearly defined boundaries which makes it very suitable as a model ecosystem in microbial ecology. Of particular importance are the transformations of C, N, and P, the solid–liquid separation properties and the functional and structural stability. A range of modern molecular methods has been used to study these communities in great detail including single cell microbiology, various -omics methods, flux analyses, and modeling making this one of the best studied microbial ecosystems so far. Recently, an EBPR core microbiome has been described and we present in this article some highlights and show how this complex microbial community can be used as model ecosystem in environmental biotechnology. [ABSTRACT FROM AUTHOR]

Objective: This study investigated the effect of commuter cycling on cardiorespiratory fitness in men and women. Secondary outcomes included body fat and blood pressure measurements. Methods: In February 2009, 48 volunteers from the Island of Funen, Denmark were randomly assigned to either “no change in lifestyle” or to “minimum 20 minutes of daily commuter cycling during 8 weeks”. Maximal oxygen uptake was assessed directly during a progressive cycle-ergometer-test, sum of skinfolds was used as an estimate of body fat, and blood pressure was measured using a Dinamap monitor. Measurements were obtained at baseline and at the conclusion of the 8-week intervention program. Results: Six cyclists dropped out. After 8 weeks O2max (mLO2 min−1) and cardiorespiratory fitness (mLO2 kg−1 min−1) were significantly improved in the cycling group when compared to the control group. Delta change in O2max between groups equaled 206mLO2 min−1 (p=0.005) and delta change in cardiorespiratory fitness equaled 2.6mLO2 kg−1 min−1 (p=0.003). Body fatness changed more noticeable (p=0.026) in the cyclists (−12.3±7.3mm) than in the controls (−0.2±7.1mm). Conclusions: O2max and cardiorespiratory fitness were significantly improved and body fat reduced as a result of people engaging in 8 weeks of commuter cycling. [Copyright &y& Elsevier]

Abstract: The microbial populations in 25 full-scale activated sludge wastewater treatment plants with enhanced biological phosphorus removal (EBPR plants) have been intensively studied over several years. Most of the important bacterial groups involved in nitrification, denitrification, biological P removal, fermentation, and hydrolysis have been identified and quantified using quantitative culture-independent molecular methods. Surprisingly, a limited number of core species was present in all plants, constituting on average approx. 80% of the entire communities in the plants, showing that the microbial populations in EBPR plants are rather similar and not very diverse, as sometimes suggested. By focusing on these organisms it is possible to make a comprehensive ecosystem model, where many important aspects in relation to microbial ecosystems and wastewater treatment can be investigated. We have reviewed the current knowledge about these microorganisms with focus on key ecophysiological factors and combined this into a conceptual ecosystem model for EBPR plants. It includes the major pathways of carbon flow with specific organic substances, the dominant populations involved in the transformations, interspecies interactions, and the key factors controlling their presence and activity. We believe that the EBPR process is a perfect model system for studies of microbial ecology in water engineering systems and that this conceptual model can be used for proposing and testing theories based on microbial ecosystem theories, for the development of new and improved quantitative ecosystem models and is beneficial for future design and management of wastewater treatment systems. [ABSTRACT FROM AUTHOR]

• Fifty Danish freshwater streams were studied using metabarcoding. • Bacterial, archaeal and eukaryotic communities were studied simultaneously. • Ecological quality was reflected across all three domains of life. • Potential indicator organisms were found in all three domains of life. • Metabarcoding shows potential for inclusion of new organisms in water quality assessments. Assessment of ecological quality in streaming surface water is often based on different Biological Quality Elements (BQE) such as plants, fish or invertebrates. Conventional stream-water quality assessment based on invertebrates as BQE relies on taxonomic expertise, which is costly and time consuming. Next-generation sequencing approaches for high-throughput analyses of diverse ecosystems are increasingly used for environmental monitoring and holds a great potential for application in stream-water quality assessments. This approach is to some extent hampered by the currently available reference databases representing freshwater invertebrates. In the present study we apply metabarcoding simultaneously targeting the 16S (prokaryotes) and 18S (eukaryotes) rRNA genes to capture a snapshot of the ecosystem composition across the three domains of life. Results based on the analysis of 50 selected Danish streams showed that the combined, as well as the domain-specific profiles can separate the samples into their respective ecological quality categories as reflected by the parallel conventional assessment based on macroinvertebrates as BQE. Furthermore, it was possible to suggest potential indicator organisms, from all three domains, which correlated specifically to the conventional data e.g. organisms with a strong correlation to ecological status across all categories. The results clearly showed that community structure in all three domains of life reflect the ecological status of the sample location. Hence, when applying a molecular approach for water-quality assessment we are not limited to the composition of visible BQE, such as macroinvertebrates. The microbial community composition in the streams may often capture an even better and more comprehensive and sensitive snapshot of the ecological quality of stream waters. [ABSTRACT FROM AUTHOR]

[Display omitted] • Changing from famine to feast/famine increased removal rates of MPs by up to 6600%. • An increase in DNA related reaction constants was reached for 24 micropollutants. • 25–60 days of optimization was required for maximum reaction rate constants. • 70 days of genomic dynamics were characterized in a feast-famine MBBR. • 88 OTUs were significantly correlated with degradation of single micropollutants. Feast-famine moving bed biofilm reactors (MBBRs) have shown high potential for removing organic micropollutants from wastewater. However, the relationship between biofilm community during feast-famine adaptation and micropollutant removal is yet unclear. In this study, we determined the biotransformation kinetics of 36 micropollutants and characterized the microbial communities in an MBBR during a 71-day adaptation period of feast-famine regime (raw/effluent wastewater). The feast-famine regime significantly changed the biodegradation rate constants (k) of 24 micropollutants in different ways: 66 times enhanced degradation for propranolol, while more than 10 times for atenolol, metoprolol, tramadol and venlafaxine, less than 2.8 times for losartan, iomeprol and iohexol were detected. 25–60 days of adaptation time was needed to reach the maximum k. Biofilm accumulated during the adaptation, but the k DNA (k relative to the biofilm with DNA concentration as a proxy for k biomass) of most micropollutants (except propranolol, metoprolol and venlafaxine) declined. This might indicate that the proliferation of potential degraders for micropollutants was slower than other microorganisms under the feast-famine regime. The microbial community changed significantly during the first 8 days of operation, followed by a relatively steady evolution towards the enrichment of nitrifiers until day 71. A multivariate statistical correlation analysis revealed that the development of occurrence of 88 individual taxonomic groups were found to exhibit a significant positive correlation to the k DNA of micropollutants (p < 0.05, r > 0.5), which represent potential biomarkers linking to biotransformation of micropollutants. These results fill the knowledge gaps between dynamics of biofilm communities and micropollutant removal in the feast-famine regime, which is essential for designing highly efficient MBBR. [ABSTRACT FROM AUTHOR]

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. • The exposure levels and the microbial compositions differed by work task. • Serum levels of CRP and SAA differed by exposure level and work tasks. • Stenotrophomonas maltophilia and Wallemia spp. were associated with high exposure. • Shannon-Wiener indices did not differ by work tasks. • The greenhouse workers were exposed to only few human pathogens. [ABSTRACT FROM AUTHOR]

• Substantial enhancement of methanogenic biofilm activity at short HRT. • Planktonic hydrogenotrophic methanogens restricted biofilm growth at long HRT. • Acetate accumulation at short HRT due to increased homoacetogenesis. • Selected hydrogenotrophic biofilm species independent of HRT and sludge source. Biomethanation exploits the ability of methanogenic archaea to convert CO 2 and renewable H 2 from electrolysis to biomethane. Biofilm reactors are promising for biomethanation scale-up due to high CH 4 productivity and low energy input for H 2 gas-liquid mass transfer. Effects of operational conditions on biofilm dynamics remain largely uncharacterized but may increase reactor potentials further. This study investigated the effect of hydraulic retention time (HRT) on methanogenic biofilm activity and composition. Commercial carriers floating in liquid were exposed to H 2 /CO 2 for 87 days with the liquid phase being subject to either 18 hours, 10 days, or 20 days HRT. Methanogenic biofilms were dominated by hydrogenotrophic methanogens, but biofilm CH 4 productivity was enhanced at 18 hours HRT due to wash-out of competing planktonic species, which otherwise hampered proliferation of biofilm biomass at long HRT. It is suggested that high-rate biofilm reactors can increase methanogenic biofilm activity by minimizing the liquid's H 2 exposure. [ABSTRACT FROM AUTHOR]

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., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Introduction: Crohn's Disease (CD) is characterized by chronic intestinal inflammation and dysbiosis. This study aimed to investigate the effects of a plant-based diet (PBD) on gut microbiota composition and inflammation in CD patients and assess the utility of trn L gene sequencing for monitoring dietary adherence., Methods: Fourteen CD patients participated in a 12-week PBD intervention. Dietary adherence was monitored through self-reported food diaries and trnL sequencing, which detects plant residues in fecal samples. Gut microbiota was analyzed using 16S rRNA sequencing, and fecal calprotectin levels were measured as an indicator of intestinal inflammation., Results: Trn L sequencing identified 55 plant genera in fecal samples, compared to 41 reported in food diaries, highlighting its accuracy in assessing plant residue diversity. By week 4, participants demonstrated a 1.4-fold increase in plant intake, correlating with a significant increase in microbial diversity. Key genera associated with gut health, such as Faecalibacterium and Bacteroides , increased in abundance. Additionally, fecal calprotectin levels decreased from 472 mg/kg at baseline to 207 mg/kg at week 12, indicating reduced intestinal inflammation., Discussion: A PBD positively influenced gut microbiota composition and decreased intestinal inflammation in CD patients. The study also demonstrated that trn L sequencing is an effective tool for assessing dietary adherence in clinical settings, offering a more objective measure than self-reported food diaries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Østergaard, Cetin, Rasmussen, Lærke, Holst, Lauridsen and Nielsen.)

This study explores the genetic landscape of nitrous oxide (N 2 O) 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 N 2 O 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 N 2 O-reducing organisms in WWTPs, highlighting their potential as N 2 O 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 N 2 O reducers and their potential as N 2 O 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., Competing Interests: The authors declare no conflict of interest.

European hedgehogs ( Erinaceus europaeus ) inhabit most of Denmark, except for a few smaller islands. Research from other European countries has shown that the hedgehog populations are in decline. The exposure to chemicals might contribute to this development, although their role is currently unknown. Our research studied the occurrence of 19 selected pesticides in the Danish hedgehog population as well as factors potentially explaining the levels of chemicals detected. We analysed 115 liver samples obtained from dead hedgehogs in 2016 for seven rodenticides, four insecticides and eight herbicides commonly used in Denmark at the time of sampling, applying a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. Detection frequencies varied between 0.9% for fluroxypyr and trans-permethrin and 79% for bromadiolone. Rodenticides, insecticides and herbicides were detected in 84, 43, and 50% of the samples, respectively. The compounds most frequently detected included the insecticide imidacloprid (35%), the herbicide metamitron (29%) and the rodenticide bromadiolone (79%). Individual concentrations varied between non-detected to >2 μg/g. A total of 79% of the 115 hedgehogs contained more than one detectable pesticide, with up to nine of the 19 compounds detected in one individual. The detection frequencies were found to differ significantly between the Eastern and Western part of Denmark for difenacoum, difethialone and imidacloprid. However, no associations were found with sex, age, habitat type or the prevalence of mecC -MRSA and endoparasites in the hedgehogs tested. Whether or not the pesticide levels detected carry a health risk for the hedgehogs remains unknown as no adverse effect levels have yet been established for European hedgehogs for single compounds or pesticide mixtures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Rasmussen, Roslev, Nielsen, Pertoldi and Vorkamp.)

Manure management is a significant source of global methane emissions, and there is an increased interest in understanding and predicting emissions. The hydrolysis rate of manure organic matter is critical for understanding and predicting methane emissions. We estimated hydrolysis rate constants of crude protein, fibers, and lipids and used the Arrhenius equation to describe its dependency on temperature. Simultaneously, measurements of methane emission, 13/12 C isotope ratios, and methanogen community were conducted. This was achieved by incubating fresh pig manure without inoculum at 10°C, 15°C, 20°C, and 25°C for 85 days in a lab-scale setup. Hydrolysis of hemicellulose and cellulose increased more with temperature than crude protein, but still, hydrolysis rate of crude protein was highest at all temperatures. Results suggested that crude protein consisted of multiple substrate groups displaying large differences in degradability. Lipids and lignin were not hydrolyzed during incubations. Cumulative methane emissions were 7.13 ± 2.69, 24.6 ± 8.00, 66.7 ± 4.8, and 105.7 ± 7.14 g CH4 kg VS -1 at 10°C, 15°C, 20°C, and 25°C, respectively, and methanogenic community shifted from Methanosphaera toward Methanocorpusculum over time and more quickly at higher temperatures. This study provides important parameter estimates and dependencies on temperature, which is important in mechanistic methane emission models. Further work should focus on characterizing quickly degradable substrate pools in the manure organic matter as they might be the main carbon source of methane emission from manure management., (© 2023 The Authors. Journal of Environmental Quality published by Wiley Periodicals LLC on behalf of American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.)

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to β-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.

Synthetic plastics, like polyethylene terephthalate (PET), have become an essential part of modern life. Many of these products are remarkably persistent in the environment, and the accumulation in the environment is recognised as a major threat. Therefore, an increasing interest has been focusing on the screening for organisms able to degrade and assimilate the plastic. Ideonella sakaiensis originally isolated from a plastisphere has been reported as a bacterium that was solely thriving on the degradation on PET films. The processes affected by the presence of PET and its monomeric substances terephthalic acid, ethylene glycol, ethyl glycolate, and sodium glyoxylate monohydrate were elucidated by analysis of differential protein expression. The exposure of PET and its monomers induced the MHETase and affect two major pathways: the TCA cycle and the β-oxidation pathway. The increased expression of proteins directly or indirectly involved in these pathways suggests their underlying importance in the degradation of PET by I. sakaiensis since these proteins are mechanistically supporting the enzymes involved in the degradation of PET and its monomers., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Collembola are a group of globally distributed microarthropods that can tolerate low temperature and are active in extremely cold environments. While it is well known that animal diets can shape their microbiota, the microbiota of soil animals is not well described, particularly for animals with limited food resources, such as Collembola active in winter at low temperatures. In this study, we explored the effects of three different food sources; corn litter (agriculture grain residuals), Mongolian oak litter (natural plant residuals), and yeast (common food for Collembola culture), on the microbiota of a winter-active Collembola species, Desoria ruseki . We found that microbial diversity and community composition of the Collembola were strongly altered after feeding with different food sources for 30 days. Collembola individuals fed on corn litter harbored the highest bacterial richness and were dominated by a representative of Microbacteriaceae . In contrast, those fed on yeast exhibited the lowest bacterial richness and were primarily colonized by Pseudomonas . The microbial communities associated with the winter-active Collembola differed significantly from those observed in the food. Collembola nutrient turnover also differed when cultured with different food sources, as indicated by the C and N stable isotopic signatures. Our study highlights microbial associations with stable isotopic enrichments of the host. Specifically, the Arthrobacter was positively correlated with δ 13 C enrichment in the host. Representatives of Microbacteriaceae , Micrococcaceae , TM7a, Devosia , and Rathayibacter were positively correlated with δ 15 N enrichment of the host. Our study indicates that food sources are major determinants for Collembola microbiota that simultaneously alter consumers' isotopic niches, thereby improving our understanding of the roles played by host-microbiota interactions in sustaining soil biodiversity during the winter., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hao, de Jonge, Zhu, Feng, Zhang, Chen, Wu and Nielsen.)

Background: Prebiotics are known to have a positive impact on fish health and growth rate, and β-glucans are among the most used prebiotics on the market. In this study, rainbow trout (Oncorhynchus mykiss) were treated with a β-1,3;1,6-glucan dietary supplement (at a dose of 0 g, 1 g, 10 g, and 50 g β-glucan per kg of feed). After 6 weeks, the effect of the β-glucan was evaluated by determining the changes in the microbiota and the blood serum metabolites in the fish. The impact of β-glucan on the immune system was evaluated through a challenge experiment with the bacterial fish pathogen Yersinia ruckeri., Results: The microbiota showed a significant change in terms of composition following β-glucan treatment, notably an increase in the relative abundance of members of the genus Aurantimicrobium, associated with a decreased abundance of the genera Carnobacterium and Deefgea. Furthermore, analysis of more than 200 metabolites revealed that the relative levels of 53 metabolites, in particular compounds related to phosphatidylcholines, were up- or downregulated in response to the dietary supplementation, this included the amino acid alanine that was significantly upregulated in the fish that had received the highest dose of β-glucan. Meanwhile, no strong effect could be detected on the resistance of the fish to the bacterial infection., Conclusions: The present study illustrates the ability of β-glucans to modify the gut microbiota of fish, resulting in alteration of the metabolome and affecting fish health through the lipidome of rainbow trout., (© 2022. The Author(s).)

Tetrabromobisphenol A (TBBPA) is the most used flame retardant worldwide and has become a threat to aquatic ecosystems. Previous research into the degradation of this micropollutant in anaerobic bioreactors has suggested several identities of putative TBBPA degraders. However, the organisms actively degrading TBBPA under in situ conditions have so far not been identified. Protein-stable isotope probing (protein-SIP) has become a cutting-edge technique in microbial ecology for enabling the link between identity and function under in situ conditions. Therefore, it was hypothesized that combining protein-based stable isotope probing with metagenomics could be used to identify and provide genomic insight into the TBBPA-degrading organisms. The identified 13 C-labelled peptides were found to belong to organisms affiliated to Phytobacter, Clostridium, Sporolactobacillus, and Klebsilla genera. The functional classification of identified labelled peptides revealed that TBBPA is not only transformed by cometabolic reactions, but also assimilated into the biomass. By application of the proteogenomics with labelled micropollutants (protein-SIP) and metagenome-assembled genomes, it was possible to extend the current perspective of the diversity of TBBPA degraders in wastewater and predict putative TBBPA degradation pathways. The study provides a link to the active TBBPA degraders and which organisms to favor for optimized biodegradation., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The gut microbiome plays a critical role in many aspects of host life, and the microbial community composition is heavily influenced by the prevailing conditions in the gut environment. Community composition has been suggested to have large implications for conservation efforts, and gut health has become of interest for optimizing animal care in captivity. In this study, we explore the gut microbiome of a wide range of animals in the context of conservation biology. The composition of the gut microbial community of 54 mammalian animal species was investigated using 16S rRNA gene amplicon sequencing. The composition of the gut microbiota clearly reflects diet and the structure of the gastrointestinal system, and it is to a certain degree more similar between closely related animals. Specific clusters of taxa were observed across animals of the same species, diet, and gut morphology. The microbiota retained regardless of captivity status is hypothesized to cover important symbiotic relationships with the host, while the remaining part reflects the artificial living conditions and can therefore be used as a future tool for conservation biologists. For five animal species (giraffes, horses, baboons, elephants, and zebras), it was possible to compare the microbiota of wild and captive individuals. Differences were observed in the proportion of microbiota detected between wild and captive specimens of the same animal species. We propose that the gut microbiota harbours important species, which can potentially serve as indicators for the well-being of the animal and the effect of living in captivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Jonge, Carlsen, Christensen, Pertoldi and Nielsen.)

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth's diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H 2 ), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C 1 -carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F 420 H 2 re-oxidation) as found for methylotrophic methanogenesis., (© 2021. The Author(s).)

The enzymatic hydrolysis of lignocellulosic polymers is generally considered the rate-limiting step to methane production in anaerobic digestion of lignocellulosic biomass. The present study aimed to investigate how the hydrolytic microbial communities of three different types of anaerobic digesters adapted to lignocellulose-rich wheat straw in continuous stirred tank reactors operated for 134 days. Cellulase and xylanase activities were monitored weekly using fluorescently-labeled model substrates and the enzymatic profiles were correlated with changes in microbial community compositions based on 16S rRNA gene amplicon sequencing to identify key species involved in lignocellulose degradation. The enzymatic activity profiles and microbial community changes revealed reactor-specific adaption of phylogenetically different hydrolytic communities. The enzymatic activities correlated significantly with changes in specific taxonomic groups, including representatives of Ruminiclostridium , Caldicoprobacter , Ruminofilibacter , Ruminococcaceae , Treponema , and Clostridia order MBA03, all of which have been linked to cellulolytic and xylanolytic activity in the literature. By identifying microorganisms with similar development as the cellulase and xylanase activities, the proposed correlation method constitutes a promising approach for deciphering essential cellulolytic and xylanolytic microbial groups for anaerobic digestion of lignocellulosic biomass., Competing Interests: NJ is employed by the company NIRAS A/S. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jensen, de Jonge, Dolriis, Kragelund, Fischer, Eskesen, Noer, Møller, Ottosen, Nielsen and Kofoed.)

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., (Copyright © 2021 American Society for Microbiology.)

We tested the effects of 18 models of robotic lawn mowers in collision with dead European hedgehogs and quantified the results into six damage categories. All models were tested on four weight classes of hedgehogs, each placed in three different positions. None of the robotic lawn mowers tested was able to detect the presence of dependent juvenile hedgehogs (<200 g) and all models had to touch the hedgehogs to detect them. Some models caused extensive damage to the hedgehog cadavers, but there were noteworthy differences in the degree of harm inflicted, with some consistently causing no damage. Our results showed that the following technical features significantly increased the safety index of the robotic lawn mowers: pivoting blades, skid plates, and front wheel drive. Based on these findings, we encourage future collaboration with the manufacturers of robotic lawn mowers to improve the safety for hedgehogs and other garden wildlife species.

Stable isotope probing (SIP) is a key tool for identifying the microorganisms catalyzing the turnover of specific substrates in the environment and to quantify their relative contributions to biogeochemical processes. However, SIP-based studies are subject to the uncertainties posed by cross-feeding, where microorganisms release isotopically labeled products, which are then used by other microorganisms, instead of incorporating the added tracer directly. Here, we introduce a SIP approach that has the potential to strongly reduce cross-feeding in complex microbial communities. In this approach, the microbial cells are exposed on a membrane filter to a continuous flow of medium containing isotopically labeled substrate. Thereby, metabolites and degradation products are constantly removed, preventing consumption of these secondary substrates. A nanoSIMS-based proof-of-concept experiment using nitrifiers in activated sludge and 13 C-bicarbonate as an activity tracer showed that Flow-SIP significantly reduces cross-feeding and thus allows distinguishing primary consumers from other members of microbial food webs.

The housefly (Musca domestica L.) lives in close association with its microbiota and its symbionts are suggested to have pivotal roles in processes such as metabolism and immune response, but it is unclear how the profound physiological changes during ontogeny affect the housefly's associated microbiota and their metabolic capabilities. The present study applies 16S rRNA gene amplicon sequencing to investigate the development of the host-associated microbiota during ontogeny. The potential for microbiota transfer between developmental stages, and the metabolic potential of these microbiota were evaluated. Representatives of Firmicutes were observed as early colonisers during the larval stages, followed by colonisation by organisms affiliating with Proteobacteria and Bacteroidetes as the flies matured into adults. Microbiota observed across all the developmental stages included Lactococcus, Lactobacillus and Enterococcus, while Weissella and Chishuiella were associated with newly hatched larvae and adults, respectively. Predictive metabolic profiling of the identified microorganisms further suggested that the microbiota and their functional profile mature alongside their host and putative host-microbe relationships are established at different stages of development. The predicted metabolic capability of the microbiota developed from primarily simple processes including carbohydrate and nucleotide metabolisms, to more complex metabolic pathways including amino acid metabolisms and processes related to signal transduction.

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to the classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.

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 β-diversity. In contrast to this, the farm from which samples were taken appeared to have the greatest effect on the bacterial β-diversity, but this trend was not observed for the fungal β-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 10 3 and 10 9 CFU/m 2 /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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Candidate phylum Saccharibacteria (former TM7) are abundant and widespread in nature, but little is known about their ecophysiology and detailed phylogeny. In this study phylogeny, morphology and ecophysiology of Saccharibacteria were investigated in activated sludge from nine wastewater treatment plants (WWTPs) from Japan and Denmark using the full-cycle 16S rRNA approach in combination with microautoradiography (MAR) and fluorescence in situ hybridization (FISH). Phylogenetic analysis showed that Saccharibacteria from all WWTPs were evenly distributed within subdivision 1 and 3 and in a distinct phylogenetic clade. Three probes were designed for the distinct saccharibacterial groups, and revealed morphotypes representing thin filaments, thick filaments and rods/cocci. MAR-FISH results showed that most probe-defined Saccharibacteria utilized glucose under aerobic-, nitrate reducing- and anaerobic conditions. Some Saccharibacteria also utilized N-acetylglucosamine, oleic acid, amino acids and butyrate, which are not predicted from available genomes so far. In addition, some filamentous Saccharibacteria exhibited β-galactosidase and lipase activities determined using a combination of enzyme-labeled fluorescence and FISH (ELF-FISH). No uptake of acetate, propionate, pyruvate, glycerol and ethanol was observed. These results indicate that Saccharibacteria is a phylogenetically diverse group and play a role in the degradation of various organic compounds as well as sugar compounds under aerobic-, nitrate reducing- and anaerobic conditions., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

'Candidatus Microthrix parvicella' is a lipid-accumulating, filamentous bacterium so far found only in activated sludge wastewater treatment plants, where it is a common causative agent of sludge separation problems. Despite attracting considerable interest, its detailed physiology is still unclear. In this study, the genome of the RN1 strain was sequenced and annotated, which facilitated the construction of a theoretical metabolic model based on available in situ and axenic experimental data. This model proposes that under anaerobic conditions, this organism accumulates preferentially long-chain fatty acids as triacylglycerols. Utilisation of trehalose and/or polyphosphate stores or partial oxidation of long-chain fatty acids may supply the energy required for anaerobic lipid uptake and storage. Comparing the genome sequence of this isolate with metagenomes from two full-scale wastewater treatment plants with enhanced biological phosphorus removal reveals high similarity, with few metabolic differences between the axenic and the dominant community 'Ca. M. parvicella' strains. Hence, the metabolic model presented in this paper could be considered generally applicable to strains in full-scale treatment systems. The genomic information obtained here will provide the basis for future research into in situ gene expression and regulation. Such information will give substantial insight into the ecophysiology of this unusual and biotechnologically important filamentous bacterium.

Ecophysiological analysis and functions of single cells in complex microbial systems can be examined by simple combinations of Fluorescence in situ hybridization (FISH) for identification with various staining techniques targeting functional phenotypes. In this chapter, we describe methods and protocols optimized for the study of extracellular enzymes, surface hydrophobicity and specific surface structures. Although primarily applied to the study of microbes in wastewater treatment (activated sludge and biofilms), the methods may also be used with minor modifications in several other ecosystems.

Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ecosystems.

The structure and function of the microbial community in a full-scale enhanced biological phosphorus removal wastewater treatment plant (WWTP; Skagen) were investigated using the full-cycle rRNA approach, combined with ecophysiological studies. A total of 87 16S rRNA gene sequences were retrieved, and 78 operational taxonomic units were identified. Novel oligonucleotide probes were designed, and quantitative fluorescence in situ hybridization revealed that six hitherto undescribed probe-defined groups within the phylum Bacteroidetes (two groups), and classes Betaproteobacteria (two groups) and Gammaproteobacteria (two groups), were relatively abundant (>1% of total biovolume) in the Skagen WWTP and 10 other full-scale WWTPs with biological P removal. The most abundant was a group of rod-shaped Bacteroidetes attached to filamentous bacteria, which is distantly related to the genus Haliscomenobacter of the family Saprospiraceae, and comprised 9-19% of the bacterial biovolume in all the WWTPs investigated. The other five probe-defined groups were found in all WWTPs, but they were less abundant (1-6%). Two groups had a glycogen-accumulating phenotype and one Dechloromonas-related group had a polyphosphate-accumulating phenotype, and they were potentially all involved in denitrification. In total, about 81% of all bacteria hybridizing with the general eubacterial probe were detected in the Skagen WWTP by using clone- or group-specific probes, indicating that most members of the microbial community had been identified.

A microbial consortia consisting of three bacteria isolated from tanning and textile wastewaters revealed high capacity to simultaneously bioaccumulate dye and Cr(VI). The identity of the bacteria were determined by 16S rRNA gene analysis to be closely related to Ochrobactrium sp., Salmonella enterica and Pseudomonas aeruginosa. Dependence of initial pH values and range of concentrations of the dye Reactive Black B (33.2-103.1 mg l(-1)) and Cr(VI) (19.9-127.6 mg l(-1)) were examined to find the effect of pH on the dye and Cr(VI) bioaccumulation. Optimal pH for growth of the consortia in media containing 35 mg l(-1) dye and 50 mg l(-1) Cr(VI) was determined to be around 8. The Cr(VI) bioaccumulation by the consortia was rapid in media containing molasses with or without reactive dye with a maximum Cr(VI) bioaccumulation yield ranging from 90% to 99% within a 2-4d period. A slightly lower yield for the dye bioaccumulation was measured with a maximum dye bioaccumulation of 80% at 59.3 mg l(-1) dye and 69.8 mg l(-1) Cr(VI). The highest specific Cr uptake value was obtained as 76.7 mg g(-1) at 117.1 mg l(-1) Cr(VI) and 50.8 mg l(-1) dye concentration. This ability to bioaccumulate dye and Cr(VI) was more efficient than the enriched sludge from which they were isolated.