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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. [ABSTRACT FROM AUTHOR]

Simple Summary: The European hedgehog (Erinaceus europaeus) is a popular visitor in gardens and recreational areas all over Europe, but hedgehog populations are declining. Research exploring the causes of the decline, including exposure to potentially harmful pollutants and metals, may provide relevant information to improve conservation initiatives to protect this species in the wild. Hedgehogs are ground-dwelling mammals, feeding on a range of different food items such as insects, slugs, snails, and earthworms but also eggs, live vertebrates, and carrion, and therefore come into close contact with pollutants present in their habitats and in their prey. This review investigated published research on the occurrence of metals and pollutants in hedgehogs and found that a vast range of different pesticides; rodenticides; persistent organic pollutants (POPs), including organochlorine compounds and brominated flame retardants (BFRs); as well as toxic heavy metals could be detected in samples from hedgehogs representing different European countries. Due to their ecology, combined with the opportunity to apply non-invasive sampling techniques through the collection of spines as sampling material, we suggest that the European hedgehog is a relevant bioindicator species for monitoring the exposure of omnivorous terrestrial wildlife to potential toxicants in urban and rural environments. 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. [ABSTRACT FROM AUTHOR]

The biogas produced through anaerobic digestion (AD) of renewable feedstocks is one of the promising alternatives to replace fossil-derived energy. Even though lignocellulosic biomass is the most abundant biomass on earth, only a small fraction is being used towards resources recovery, leaving a great potential unexploited. In this study, the combination of state-of-art genomic techniques and engineered systems were used to further advance the knowledge on biogas production from lignocellulosic-rich residues and the microbiome involved in the anaerobic digestion hereof. A long-term adapted anaerobic microbiome capable of degrading wheat straw as the sole substrate was investigated using protein stable isotope probing (protein-SIP). The results indicated that a diverse microbial community, primarily composed of Firmicutes and Methanogens, played crucial roles in cellulose degradation and methane production. Notably, Defluviitoga tunisiensis, Syntrophothermus lipocalidus, and Pelobacter carbinolicus were identified as direct metabolizers of cellulose, while Dehalobacterium assimilated labelled carbon through cross-feeding. This study provides direct evidence of primary cellulose degraders and sheds light on their genomic composition. By harnessing the potential of lignocellulosic biomass and understanding the microbial communities involved, we can promote sustainable biogas production, contributing to energy security and environmental preservation. [ABSTRACT FROM AUTHOR]

The recent development of techniques to sequence ancient DNA has provided valuable insights into the civilisations that came before us. However, the full potential of these methods has yet to be realised. We extracted ancient DNA from a recently exposed fracture surface of a clay brick deriving from the palace of king Ashurnasirpal II (883–859 BCE) in Nimrud, Iraq. We detected 34 unique taxonomic groups of plants. With this research we have made the pioneering discovery that ancient DNA, effectively protected from contamination inside a mass of clay, can successfully be extracted from a 2900-year-old clay brick. We encourage future research into this subject, as the scientific prospects for this approach are substantial, potentially leading to a deeper understanding of ancient and lost civilisations. [ABSTRACT FROM AUTHOR]

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 winteractive 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 winteractive 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 d13C enrichment in the host. Representatives of Microbacteriaceae, Micrococcaceae, TM7a, Devosia, and Rathayibacter were positively correlated with d15N 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Microalgae comprise a phylogenetically very diverse group of photosynthetic unicellular pro‐ and eukaryotic organisms growing in marine and other aquatic environments. While they are well explored for the generation of biofuels, their potential as a source of antimicrobial and prebiotic substances have recently received increasing interest. Within this framework, microalgae may offer solutions to the societal challenge we face, concerning the lack of antibiotics treating the growing level of antimicrobial resistant bacteria and fungi in clinical settings. While the vast majority of microalgae and their associated microbiota remain unstudied, they may be a fascinating and rewarding source for novel and more sustainable antimicrobials and alternative molecules and compounds. In this review, we present an overview of the current knowledge on health benefits of microalgae and their associated microbiota. Finally, we describe remaining issues and limitation, and suggest several promising research potentials that should be given attention. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

With an accelerating negative impact of anthropogenic actions on natural ecosystems, non-invasive biodiversity assessments are becoming increasingly crucial. As a consequence, the interest in the application of environmental DNA (eDNA) survey techniques has increased. The use of eDNA extracted from faeces from generalist predators, have recently been described as "biodiversity capsules" and suggested as a complementary tool for improving current biodiversity assessments. In this study, using faecal samples from two generalist omnivore species, the Eurasian badger and the red fox, we evaluated the applicability of eDNA metabarcoding in determining dietary composition, compared to macroscopic diet identification techniques. Subsequently, we used the dietary information obtained to assess its contribution to biodiversity assessments. Compared to classic macroscopic techniques, we found that eDNA metabarcoding detected more taxa, at higher taxonomic resolution, and proved to be an important technique to verify the species identification of the predator from field collected faeces. Furthermore, we showed how dietary analyses complemented field observations in describing biodiversity by identifying consumed flora and fauna that went unnoticed during field observations. While diet analysis approaches could not substitute field observations entirely, we suggest that their integration with other methods might overcome intrinsic limitations of single techniques in future biodiversity surveys. [ABSTRACT FROM AUTHOR]

The Eurasian otter (Lutra lutra) is a semi-aquatic predator that lives in a wide range of aquatic habitats. Knowledge of the diet of predators is essential for the management and conservation of the species and their habitats. We assessed the diet of otters in fifty-three faecal samples (spraints) by morphological analysis of undigested prey remains and by DNA metabarcoding approaches with two different blocking primers. The blocking primers were shown to be similarly effective in repressing host DNA, improving amplification of non-host taxa. The analyses were conducted to compare the accuracy of the two methods. The prey items were categorized to the highest possible taxonomic resolution, and relative frequency of occurrence was applied in order to compare the morphological and DNA results. The bulk score for difference prey items in the spraints was based on volume estimates in macroscopic observations. The two methods had an overlap of identified prey species ranging from 69.9 to 72.9% (depending on the kind of primers) meaning that a large portion of prey items found by one method was replaced by other prey items in the other method and that most prey items found through DNA-metabarcoding are considered new when compared with the morphological analysis. Therefore, applying both morphological and DNA analysis is crucial in order to determine the diet of otters and, possibly, other predatory species. [ABSTRACT FROM AUTHOR]

Re-introduction of large herbivores is increasingly used as a tool in nature management and for restoration of more biodiverse habitats. This study investigated the diet of recently introduced European bison, Bison bonasus, in a forest habitat in Bornholm, Denmark, with the purpose of evaluating its adaptation to the new habitat and assessing its potential for facilitating development towards a more open and species-rich forest. Metabarcoding of 39 bison dung samples collected during June–August identified 71 plant taxa belonging to 36 families as diet objects, comprising 56% forbs, 20% trees, 17% graminoids and 7% shrubs. The broad composition of the diet shows the capacity of the bison to adjust to new habitats and exploit a variety of different habitats when foraging, including meadows and other wet areas. Among the most frequently consumed plant taxa were the shrub Rubus idaeus, a number of coarse grass species and most tree species found in the habitat. Rubus idaeus were by far the most abundant taxa, constituting 44% of all the DNA sequences. The results suggest that foraging by bison can contribute towards limiting domination by understory shrubs and high, coarse grasses. Over an extended time period, foraging by bison is expected to promote development towards a lower and more open understory and herbaceous layer in the forest habitats. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objectives: Low genetic diversity can lead to reduced average fitness in a population or even extinction. Preserving genetic connectivity across fragmented landscapes is therefore vital to counteract the negative consequences of genetic drift and inbreeding. This study aimed to assess the genetic composition and consequently the conservation status of a nationwide sample of European hedgehogs (Erinaceus europaeus) in Denmark. Methods: We applied an adaptation of the genotyping by sequencing (GBS) technique to 178 individuals from six geographically distinct populations. We used a Bayesian clustering method to subdivide individuals into genetically distinct populations. We estimated individual observed (iHO), observed (HO), and unbiased expected (uHE) heterozygosity, inbreeding coefficient (FIS), percentage of polymorphic loci (P%) and tested for deviations from Hardy-Weinberg equilibrium (HWE). We used linear models to test for potential anthropogenic effects on the genetic variability of hedgehogs with iHO, uHE, P% and FIS as response variables, and assessed the demographic history of the population. Results: The Danish hedgehog population is composed of three genetic clusters. We found a mean P% of 54.44–94.71, a mean uHE of 0.126–0.318 and a mean HO of 0.124–0.293 in the six populations. The FIS was found to be significantly positive for three of the six populations. We detected a large heterogeneity of iHO values within populations, which can be due to inbreeding and/or fragmentation. FIS values decreased with increasing farmland density, but there was no significant association with human population or road density. Conclusions: We found a low level of genetic variability and evidence for genetic substructure and low effective population size, which are all consequences of habitat fragmentation. We failed to detect signs of a recent population bottleneck or population increase or decline. However, because the test only identifies recent changes in population size, we cannot reject the possibility of a longer-term decline in the Danish hedgehog population. [ABSTRACT FROM AUTHOR]

Administrating antibiotics to young piglets may have short- and long-term consequences on the gut microbiota. We hypothesised that these consequences may be alleviated by concurrent probiotic administration. The study objective was to investigate the effect of administrating gentamicin and a mixture of Bacillus (B.) licheniformis, B. subtilis and B. amyloliquefaeceans spores on the gut microbiota of piglets pre- and post-weaning. Twenty-four sows and their litters were randomly allocated to four treatment groups receiving; a) Bacillus spore mixture (six B. subtilis, two B. amyloliquefaeceans, and one B. licheniformis) fed to sows and piglets (PRO); b) gentamicin (5 mg per day) administered to piglets on day 4, 5, and 6 of age (AB); c) Bacillus spore mixture fed to sows and piglets, and gentamicin to piglets (PRO+AB); or d) no administration of probiotics or antibiotics (CTRL). Faecal and digesta samples were collected repeatedly during the study. Selected samples were subjected to 16S rRNA gene sequencing, culture counts, and organic acid, biogenic amine and tissue gene expression analysis. Treatment had a significant effect on the faecal microbial community composition on day 28 and 42, and colonic community on day 28. Faecal species richness (observed and estimated) and Shannon index, and colonic species richness, were higher in AB compared to PRO piglets on day 28, and were not significantly different from day 42. PRO piglets had the highest faecal concentration of iso-butyric acid on day 7 and a higher butyric acid concentration compared to CTRL piglets. We conclude that gentamicin and Bacillus spores influence the gut microbial diversity of piglets, although administration of gentamicin did not result in dysbiosis as hypothesised. [ABSTRACT FROM AUTHOR]

The housefly feeds and reproduces in animal manure and decaying organic substances and thus lives in intimate association with various microorganisms including human pathogens. In order to understand the variation and association between bacteria and the housefly, we used 16S rRNA gene amplicon sequencing to describe bacterial communities of 90 individual houseflies collected within and between ten dairy farms in Denmark. Analysis of gene sequences showed that the most abundant classes of bacteria found across all sites included Bacilli, Clostridia, Actinobacteria, Flavobacteria, and all classes of Proteobacteria and at the genus level the most abundant genera included Corynebacterium, Lactobacillus, Staphylococcus, Vagococcus, Weissella, Lactococcus, and Aerococcus. Comparison of the microbiota of houseflies revealed a highly diverse microbiota compared to other insect species and with most variation in species richness and diversity found between individuals, but not locations. Our study is the first in-depth amplicon sequencing study of the housefly microbiota, and collectively shows that the microbiota of single houseflies is highly diverse and differs between individuals likely to reflect the lifestyle of the housefly. We suggest that these results should be taken into account when addressing the transmission of pathogens by the housefly and assessing the vector competence variation under natural conditions. [ABSTRACT FROM AUTHOR]

Effective and stable nitrogen removal from wastewater requires abundant and active denitrifying populations. In this study, a one-year investigation of the population dynamics of phylogenetic groups known to harbor nitrate reducers was conducted in three municipal wastewater treatment plants (WWTPs). The bacterial community composition was determined by amplicon sequencing of the 16S rRNA gene, and putative nitrate reducers were identified by sequencingnarGandnapAgenes. Fluorescencein situhybridization with oligonucleotide probes targeting known nitrate reducers in wastewater revealed that certain bacteria predominated in the WWTPs:Curvibacter-related bacteria,Comamonadaceae,Azoarcus, Thauera, Dechloromonas, andCandidatusAccumulibacter withinRhodocyclaceae. The data showed high diversity in the nitrate-reducing community and a large degree of redundancy, with a relatively stable core group of bacteria in each plant that ensured small yearly variation in nitrate reduction rates. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 melanogaster at 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. [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]

The Microbial Database for Activated Sludge (MiDAS) field guide is a freely available online resource linking the identity of abundant and process critical microorganisms in activated sludge wastewater treatment systems to available data related to their functional importance. Phenotypic properties of some of these genera are described, but most are known only from sequence data. The MiDAS taxonomy is a manual curation of the SILVA taxonomy that proposes a name for all genus-level taxa observed to be abundant by large-scale 16 S rRNA gene amplicon sequencing of full-scale activated sludge communities. The taxonomy can be used to classify unknown sequences, and the online MiDAS field guide links the identity to the available information about their morphology, diversity, physiology and distribution. The use of a common taxonomy across the field will provide a solid foundation for the study of microbial ecology of the activated sludge process and related treatment processes. The online MiDAS field guide is a collaborative workspace intended to facilitate a better understanding of the ecology of activated sludge and related treatment processes--knowledge that will be an invaluable resource for the optimal design and operation of these systems. Database URL: http://www.midasfieldguide.org [ABSTRACT FROM AUTHOR]

In this study, a temperature-sensitive fabric with antibacterial properties was prepared by the formation of silver nanoparticles (AgNPs) on nonwoven poly (propylene) (PP) grafted with poly (N-iso-propylacrylamide) (PNIPAAm-PP). First, PNIPAAm was grafted onto corona-treated nonwoven PP. Afterwards, silver nanoparticles were synthesized on the temperature-sensitive hydrogel layer grafted to the surface of nonwoven PP by the reduction of silver ions (Ag+). Fourier transform infra-red spectroscopy confirmed the presence of PNIPAAm on the nonwoven PP. scanning electron microscopy-energy dispersive X-ray spectroscopy was used to investigate surface morphology and the presence of silver particles in the samples. Inductively coupled-plasma atomic emission spectroscopy revealed that the Ag content in Ag-functionalized PNIPAAm-PP was significantly higher than Ag-functionalized corona-treated PP with the same concentration of silver solution. Moreover, the results of the swelling rate experiment confirmed that PNIPAAm-PP maintained temperature-sensitive properties after functionalizing with Ag. The results showed that the formation of AgNPs with enhancement in antibacterial property was possible onto PNIPAAm-PP. [ABSTRACT FROM AUTHOR]

Microbial communities are typically characterized by conditions of nutrient limitation so the availability of the resources is likely a key factor in the niche differentiation across all species and in the regulation of the community structure. In this study we have investigated whether four species exhibit any in situ short-term changes in substrate uptake pattern when exposed to variations in substrate and growth conditions. Microautoradiography was combined with fluorescence in situ hybridization to investigate in situ cell-specific substrate uptake profiles of four probe-defined coexisting species in a wastewater treatment plant with enhanced biological phosphorus removal. These were the filamentous ' Candidatus Microthrix' and Caldilinea (type 0803), the polyphosphate-accumulating organism ' Candidatus Accumulibacter', and the denitrifying Azoarcus. The experimental conditions mimicked the conditions potentially encountered in the respective environment (starvation, high/low substrate concentration, induction with specific substrates, and single/multiple substrates). The results showed that each probe-defined species exhibited very distinct and constant substrate uptake profile in time and space, which hardly changed under any of the conditions tested. Such niche partitioning implies that a significant change in substrate composition will be reflected in a changed community structure rather than the substrate uptake response from the different species. [ABSTRACT FROM AUTHOR]

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to 'Candidatus Accumulibacter phosphatis' is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by 'Candidatus Accumulibacter phosphatis', and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation. [ABSTRACT FROM AUTHOR]

Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAOs) in full-scale enhanced biological phosphorus removal (EBPR) plants. Clone library analyses and application of MAR-FISH using newly designed probes revealed that small rods related to uncultured Halomonas within the gammaproteobacterial family Halomonadaceae were actively involved in uptake of orthophosphate. Although deeply branched in the Gammaproteobacteria, they were not targeted by the gammaproteobacterial probe (GAM42a). A part of them were also not targeted with the general bacterial probes (EUBmix). They could take up short-chain fatty acids (e.g. acetate and propionate) and ethanol under both anaerobic and aerobic conditions. Polyhydroxyalkanoate storage was observed under anaerobic conditions. There was no indication of a denitrifying capability. A survey of the occurrence of these Halomonas-PAOs in 23 full-scale EBPR plants revealed that they made up 0.5-5.7% of all bacteria in the plants, and were often in higher abundance than the well-described PAOs ' Candidatus Accumulibacter phosphatis'. This indicates a potentially important role for these uncultured Halomonas bacteria in the EBPR process in full-scale plants and we propose to name them ' Candidatus Halomonas phosphatis'. [ABSTRACT FROM AUTHOR]

The diversity of the putative polyphosphate-accumulating genus Tetrasphaera in wastewater treatment systems with enhanced biological phosphorus removal (EBPR) was investigated using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. 16S rRNA actinobacterial gene sequences were retrieved from different full-scale EBPR plants, and the sequences belonging to the genus Tetrasphaera (family Intrasporangiaceae) were found to form three clades. Quantitative FISH analyses of the communities in five full-scale EBPR plants using 10 new oligonucleotide probes were carried out. The results showed that the probe-defined Tetrasphaera displayed different morphologies and constituted up to 30% of the total biomass. It was shown that active uptake of orthophosphate and formation of polyphosphate took place in most of the probe-defined Tetrasphaera populations. However, aerobic uptake of orthophosphate only took place after uptake of certain carbon sources under anaerobic conditions and these were more diverse than hitherto assumed: amino acids, glucose, and for some also acetate. Tetrasphaera seemed to occupy a slightly different ecological niche compared with ' Candidatus Accumulibacter' contributing to a functional redundancy and stability of the EBPR process. [ABSTRACT FROM AUTHOR]

A new approach for quantification of extracellular DNA (eDNA) in mixed biofilms at microscale resolution was developed and combined with other staining techniques to assess the origin, abundance and role of eDNA in activated sludge biofilms. Most eDNA was found in close proximity to living cells in microcolonies, suggesting that most of it originated from an active secretion or alternatively, by lysis of a sub-population of cells. When the staining was combined with fluorescence in situ hybridization for identification of the microorganisms, it was found that the eDNA content varied among the different probe-defined species. The highest amount of eDNA was found in and around the microcolonies of denitrifiers belonging to the genera Curvibacter and Thauera, the ammonium-oxidizing Nitrosomonas and the nitrite-oxidizing Nitrospira. Other floc-formers also produced eDNA, although in lower amounts. The total eDNA content in activated sludge varied from 4 to 52 mg per gram volatile suspended solids in different wastewater treatment plants. Very high local concentrations within some microcolonies were found with up to approximately 300 mg of eDNA per g of organic matter. DNase digestion of activated sludge led to general floc disintegration and disruption of the microcolonies with high eDNA content, implying that eDNA was an important structural component in activated sludge biofilms. [ABSTRACT FROM AUTHOR]

The distribution of Thioploca populations was investigated in Danish fjords, brackish lakes and coastal waters. Thioploca was found in three geographically distinct populations, where biomasses reached 33.8±14.3 g wet weight m−2 (mean±SD). Mats or lawns were not formed at the sediment surfaces and Thioploca biomasses peaked 4–7 cm into the sediment and extended down to 18 cm depth. Morphology and 16S rRNA gene sequences classified all populations as Thioploca ingrica. A sequence divergence of 1.7–2.2% indicated that T. ingrica comprise at least two genotypes. Physiological analysis showed that T. ingrica accumulate nitrate in concentrations of approximately 3 mM and that bicarbonate and acetate are used as a carbon source. The presence of oxygen promoted carbon incorporation, but T. ingrica could survive up to 3 months without an external supply of nitrate or oxygen. Thioploca ingrica populations were exclusively found close to river outlets in a bioturbated sediment with separate sulphidic spots and worm burrow walls containing nitrate and oxygen. It is hypothesized that the subsurface T. ingrica have a special advantage in this heterogeneous environment using their sheath surrounding the bacterial trichomes when navigating between electron donor and acceptor. [ABSTRACT FROM AUTHOR]

Excessive growth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) can cause serious operational problems. With some filaments there may be the problem of bulking, where inadequate flocculation and settling of the biomass in the secondary clarifier results in a carryover of solids with the final treated liquid effluent. Their proliferation often encourages the development of stable foams on the surface of the reactors, and these foams may impact negatively on plant performance and operation. The availability of culture-independent molecular methods now allows us to identify many of the more common filamentous organisms encountered in WWTPs, which are phylogenetically diverse, affiliating to seven separate bacterial phyla. Furthermore, the extensive data published in the past decade on their in situ behaviour from the application of these culture-independent methods have not been summarized or reviewed critically. Hence, here, we attempt to discuss what we now know about their identity, ecophysiology and ecological niches and its practical value in better managing activated sludge processes. Some of this knowledge is already being applied to control and manage full-scale WWTPs better, and the hope is that this review will contribute towards further developments in this field of environmental microbiology. [ABSTRACT FROM AUTHOR]

Among prokaryotes, the large vacuolated marine sulphur bacteria are unique in their ability to store, transport and metabolize significant quantities of sulphur, nitrogen, phosphorus and carbon compounds. In this study, unresolved questions of metabolism, storage management and behaviour were addressed in laboratory experiments with Thioploca species collected on the continental shelf off Chile. The Thioploca cells had an aerobic metabolism with a potential oxygen uptake rate of 1760 μmol O2 per dm3 biovolume per h, equivalent to 4.4 nmol O2 per min per mg protein. When high ambient sulphide concentrations (∼200 μM) were present, a sulphide uptake of 6220±2230 μmol H2S per dm3 per h, (mean±s.e.m., n=4) was measured. This sulphide uptake rate was six times higher than the oxidation rate of elemental sulphur by oxygen or nitrate, thus indicating a rapid sulphur accumulation by Thioploca. Thioploca reduce nitrate to ammonium and we found that dinitrogen was not produced, neither through denitrification nor through anammox activity. Unexpectedly, polyphosphate storage was not detectable by microautoradiography in physiological assays or by staining and microscopy. Carbon dioxide fixation increased when nitrate and nitrite were externally available and when organic carbon was added to incubations. Sulphide addition did not increase carbon dioxide fixation, indicating that Thioploca use excess of sulphide to rapidly accumulate sulphur rather than to accelerate growth. This is interpreted as an adaptation to infrequent high sulphate reduction rates in the seabed. The physiology and behaviour of Thioploca are summarized and the adaptations to an environment, dominated by infrequent oxygen availability and periods of high sulphide abundance, are discussed.The ISME Journal (2009) 3, 647–657; doi:10.1038/ismej.2009.17; published online 5 March 2009 [ABSTRACT FROM AUTHOR]

The denitrification capacity of different phylogenetic bacterial groups was investigated on addition of different substrates in activated sludge from two nutrient-removal plants. Nitrate/nitrite consumption rates (CRs) were calculated from nitrate and nitrite biosensor, in situ measurements. The nitrate/nitrite CRs depended on the substrate added, and acetate alone or combined with other substrates yielded the highest rates (3–6 mg N gVSS−1 h−1). The nitrate CRs were similar to the nitrite CRs for most substrates tested. The structure of the active denitrifying population was investigated using heterotrophic CO2 microautoradiography (HetCO2-MAR) and FISH. Probe-defined denitrifiers appeared as specialized substrate utilizers despite acetate being preferentially used by most of them. Azoarcus and Accumulibacter abundance in the two different sludges was related to differences in their substrate-specific nitrate/nitrite CRs. Aquaspirillum-related bacteria were the most abundant potential denitrifiers ( c. 20% of biovolume); however, Accumulibacter (3–7%) and Azoarcus (2–13%) may have primarily driven denitrification by utilizing pyruvate, ethanol, and acetate. Activated sludge denitrification was potentially conducted by a diverse, versatile population including not only Betaproteobacteria ( Aquaspirillum, Thauera, Accumulibacter, and Azoarcus) but also some Alphaproteobacteria and Gammaproteobacteria, as indicated by the assimilation of 14CO2 by these probe-defined groups with a complex substrate mixture as an electron donor and nitrite as an electron acceptor in HetCO2-MAR–FISH tests. [ABSTRACT FROM AUTHOR]

The efficiency of removing unwanted biofilm from surfaces in industrial water systems was examined by fluorescence microscopy and image analysis. A quantitative assay for in situ determination of biofilm components was developed and tested on thin biofilms grown in reactors as well as real biofilms sampled from a fish processing factory. Different fluorescent dyes for in situ detection of protein, lipid and total organic matter were tested. It was possible to determine the approximate amounts, concentrations and coverage of the different components by correlating the fluorescent intensity of the biofilm components to standard solutions immobilised as a biofilm. The quantification methods were evaluated as a strategy for determining the efficiency of different disinfection/cleaning procedures, showing that quantification of these biofilm components was fast and reliable for optimisation of cleaning in place procedures. However, the approach also showed that bacterial cells, as investigated by culture-independent procedures, were killed but not removed by most disinfection procedures tested, potentially leading to surfaces which are easily recolonised. [ABSTRACT FROM AUTHOR]

Surface-associated amyloid fibrils have been described by bacteria in the family Enterbacteriaceae, but it is unknown to what extent amyloid adhesins are present in natural biofilms. In this study, amyloid adhesins were specifically stained with Thioflavin T and two conformationally specific antibodies targeting amyloid fibrils. These three independent detection methods were each combined with fluorescence in situ hybridization using fluorescently labelled oligonucleotide probes in order to link phenotype with identity. Escherichia coli mutants with and without amyloid adhesins (curli) served as controls. In biofilms from four different natural habitats, bacteria producing extracellular amyloid adhesins were identified within several phyla: Proteobacteria ( Alpha-, Beta-, Gamma- and Deltaproteobacteria), Bacteriodetes, Chloroflexi and Actinobacteria, and most likely also in other phyla. Quantification of the microorganisms producing amyloid adhesins showed that they constituted at least 5–40% of all prokaryotes present in the biofilms, depending on the habitat. Particularly in drinking water biofilms, a high number of amyloid-positive bacteria were identified. Production of amyloids was confirmed by environmental isolates belonging to the Gammaproteobacteria, Bacteriodetes, Firmicutes and Actinobacteria. The new approach is a very useful tool for further culture-independent studies in mixed microbial communities, where the abundance and diversity of bacteria expressing amyloid adhesins seems much greater than hitherto anticipated. [ABSTRACT FROM AUTHOR]

Increasing incidences of activated sludge foaming have been reported in the last decade in Danish plants treating both municipal and industrial wastewaters. In most cases, foaming is caused by the presence of Actinobacteria; branched mycolic acid-containing filaments (the Mycolata) and the unbranched Candidatus‘ Microthix parvicella’. Surveys from wastewater treatment plants revealed that the Mycolata were the dominant filamentous bacteria in the foam. Gordonia amarae-like organisms and those with the morphology of Skermania piniformis were frequently observed, and they often coexisted. Their identity was confirmed by FISH, using a new permeabilization procedure. It was not possible to identify all abundant Mycolata using existing FISH probes, which suggests the presence of currently undetectable and potentially undescribed populations. Furthermore, some Mycolata failed to give any FISH signal, although substrate uptake experiments with microautoradiography revealed that they were physiologically active. Ecophysiological studies were performed on the Mycolata identified by their morphology or FISH in both foams and mixed liquors. Large differences were seen among the Mycolata in levels of substrate assimilation and substrate uptake abilities in the presence of different electron acceptors. These differences were ascribed mainly to the presence of currently undescribed Mycolata species and/or differences in foam age. [ABSTRACT FROM AUTHOR]

Abstract: A comprehensive study of the ecophysiology of the filamentous Meganema perideroedes affiliated to the Alphaproteobacteria, possessing a “Nostocoida limicola Type II” filamentous morphology was conducted. This morphotype often causes serious bulking problems in activated sludge wastewater treatment plants, and hardly anything is known about its physiology. The study was carried out by applying a suite of in situ methods in an industrial activated sludge treatment plant with excessive growth of this species. The experiments revealed a very versatile organism able to take up a large variety of organic substrates under aerobic conditions. It had a remarkably high storage capacity forming polyhydroxyalkanoates from most substrates tested. When nitrate was present as e-acceptor, the number of substrates to be consumed by M. perideroedes was more restricted compared to aerobic conditions. With nitrite as e-acceptor, only acetate and glucose among the substrates tested could be assimilated and used for storage and possibly growth. This indicated that M. perideroedes might be able to denitrify under certain conditions, which is unusual for filamentous bacteria in activated sludge. No substrate uptake or storage was seen under anaerobic conditions. M. perideroedes was relatively hydrophobic, compared to other filamentous bacteria and microcolonies present in the sludge, indicating the presence of a hydrophobic sheath. Several excreted surface-associated exoenzymes were detected in the sludge, but M. perideroedes never showed any activity, except once after a breakdown in the production facility. This confirmed that M. perideroedes mainly grows on soluble substrates. Based on the studies of the ecophysiology of M. perideroedes, potential control strategies are suggested. [Copyright &y& Elsevier]

Local nitrification and carbon assimilation activities were studied in situ in a model biofilm to investigate carbon yields and contribution of distinct populations to these activities. Immobilized microcolonies (related to Nitrosomonas europaea/ eutropha, Nitrosomonas oligotropha, Nitrospira sp., and to other Bacteria) were incubated with [14C]-bicarbonate under different experimental conditions. Nitrifying activity was measured concomitantly with microsensors (oxygen, ammonium, nitrite, nitrate). Biofilm thin sections were subjected to fluorescence in situ hybridization (FISH), microautoradiography (MAR), and local quantification of [14C]-bicarbonate uptake (beta microimaging). Nitrifying activity and tracer assimilation were restricted to a surface layer of different thickness in the various experiments (substrate or oxygen limitation). Excess oxygen uptake under all conditions revealed heterotrophic activity fuelled by decay or excretion products during active nitrification. Depth limits and intensity of tracer incorporation profiles were in agreement with ammonia-oxidation activity (measured with microsensors), and distribution of incorporated tracer (detected with MAR). Microautoradiography revealed a sharp individual response of distinct populations in terms of in-/activity depending on the (local) environmental conditions within the biofilm. Net in situ carbon yields on N, expressed as e– equivalent ratios, varied between 0.005 and 0.018, and, thus, were in the lower range of data reported for pure cultures of nitrifiers. [ABSTRACT FROM AUTHOR]

Microautoradiography is a technique that enables direct detection of active bacteria in complex microbial systems on a single cell level. When combined with fluorescence in situ hybridization using oligonucleotide probes for identification of the microorganisms, it is possible to link key physiological features to the identity of microorganism. In this way information can be obtained about the physiology of cultured as well as not-yet cultured microorganism in biofilms and other complex ecosystems in a way that presently cannot be provided by other methods. In this small review we describe the basic procedure, the type of information that can be obtained, the most important limitations by using this approach, and some recent examples from biofilms and other complex microbial communities. [ABSTRACT FROM AUTHOR]

Summary A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe-defined filamentous bacteria directly in a complex system. The technique, QMAR-FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria ‘Candidatus Meganema perideroedes’ and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium-labelled substrates revealed an average cell-specific uptake rate of 4.1 ¥ 10-15 mol of acetate cell-1 h-1 and 3.1 ¥ 10-15 mol of acetate cell-1 h-1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe-defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (K s ) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The K s values of the ‘Candidatus Meganema perideroedes’ and the Thiothrix filamentous bacteria were determined to be 1.8 µM and 2.4 µM acetate respectively. [ABSTRACT FROM AUTHOR]

Summary Among the filamentous bacteria occasionally causing bulking problems in activated sludge treatment plants, three morphotypes with attached microbial growth are common, Eikelboom Type 0041, Type 1851 and Type 1701. A better knowledge of the phylogeny and physiology of these filamentous bacteria is necessary in order to develop control strategies for bulking. In this study we have used a combination of fluorescence in situ hybridization (FISH) and microautoradiography (MAR) to investigate the identity and in situ physiology of the Type 0041-morphotype and its attached bacteria in two wastewater treatment plants. Identification and enumeration of Type 0041 using group-specific 16S rRNA-targeted FISH probes revealed that approximately 15% of the filaments hybridized with a gene probe specific for the TM7 group, a recently recognized major lineage in the bacterial domain. All other filaments morphologically identified as Type 0041 only hybridized to the general bacterial EUB338-probe, indicating that they probably do not belong to commonly isolated bacterial phyla such as the Proteobacteria , Firmicutes , Actinobacteria and Bacteroidetes , for which group-specific probes were used. The phylogenetic heterogeneity of Type 0041 again highlights the inadequacy of a morphology-based classification system. Like the filaments, most of the attached microbial cells were not identified beyond their affiliation to the Bacteria using the group-specific FISH probes. However, several different bacterial phyla were represented in the identified fraction suggesting that the attached microorganisms are phylogenetically diverse. The study of the in situ physiology of Type 0041 using MAR-FISH revealed that both the filaments and the attached bacteria on Type 0041 were versatile in the use of organic substrates and electron acceptors. It was observed that all Type 0041 could consume glucose, but none of the filaments were able to consume acetate under any conditions tested, in... [ABSTRACT FROM AUTHOR]