Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation.

BACKGROUND: Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. RESULTS: Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. CONCLUSIONS: The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.

Nexus between the microbial diversity level and the stress tolerance within the biogas process.

To investigate whether there is a nexus between the microbial diversity level (taxonomic, functional and ecological) and the stress tolerance potential of the microbial community, the impact of different ammonium sources was evaluated. Therefore reactors adapted either to the anaerobic digestions of sugar beet silage or maize silage (SBS/MS) were supplemented with animal manure (M) or ammonium carbonate (A). The results showed that increasing concentrations of total ammonium nitrogen (TAN) were not the only reason for community changes: the bacterial community in the reactors given animal manure became more similar over time compared to the reactors given ammonium carbonate. However, this study revealed that a bacterial community with a few dominant members led to a functional more flexible archaeal community (SBS reactors) which was more stress resistant under the experimental conditions. This indicates that a higher functional diversity within a certain part of the community, in the present study the archaeal community, is one important factor for process stability due to a higher tolerance to increasing amounts of process-inhibiting metabolites such as TAN. Compared to this a bacterial community with higher amount of more evenly distributed community members combined with a more rigid archaeal community (MS reactors) showed a lower stress tolerance potential. Moreover it was observed that the disappearance of members of the phylum Cloacimonetes can be used as an indicator for an upcoming process disturbance due to increasing TAN concentrations.

Microbial communities and processes in biofilters for post-treatment of ozonated wastewater treatment plant effluent.

Ozonation is an established solution for organic micropollutant (OMP) abatement in tertiary wastewater treatment. Biofiltration is the most common process for the biological post-treatment step, which is generally required to remove undesired oxidation products from the reaction of ozone with water matrix compounds. This study comparatively investigates the effect of filter media on the removal of organic contaminants and on biofilm properties for biologically activated carbon (BAC) and anthracite biofilters. Biofilms were analysed in two pilot-scale filters that have been operated for >50,000 bed volumes as post-treatment for ozonated wastewater treatment plant effluent. In parallel, the removal performance of bulk organics and OMP, including differentiation of adsorption and biotransformation through sodium azide inhibition, were carried out in bench-scale filter columns filled with material from the pilot filters. The use of BAC instead of anthracite resulted in an improved removal of organic bulk parameters, dissolved oxygen, and OMP. The OMP removal observed in the BAC filter but not in the anthracite filter was based on adsorption for most of the investigated compounds. For valsartan, however, biotransformation was found to be the dominant pathway, indicating that conditions for biotransformation of certain OMP are better on BAC than on anthracite. Adenosine triphosphate analyses in the media-attached biofilms of the pilot filters showed that biomass concentrations in the BAC filter were significantly higher than in the anthracite filter. The microbial communities (16S rRNA gene sequencing) appeared to be similar with respect to the types of organisms occurring on both filter materials. Alpha diversity also exhibited little variation between filter media. Beta diversity analysis, however, revealed that filter media and bed depth substantially influenced the biofilm composition. In practice, the impact of filter media on biofilm properties and biotransformation processes should be considered for the design of biofilters.

Characterization of phage vB_EcoS-EE09 infecting E. coli DSM613 Isolated from Wastewater Treatment Plant Effluent and Comparative Proteomics of the Infected and Non-Infected Host.

Phages influence microbial communities, can be applied in phage therapy, or may serve as bioindicators, e.g., in (waste)water management. We here characterized the Escherichia phage vB_EcoS-EE09 isolated from an urban wastewater treatment plant effluent. Phage vB_EcoS-EE09 belongs to the genus Dhillonvirus, class Caudoviricetes. It has an icosahedral capsid with a long non-contractile tail and a dsDNA genome with an approximate size of 44 kb and a 54.6% GC content. Phage vB_EcoS-EE09 infected 12 out of the 17 E. coli strains tested. We identified 16 structural phage proteins, including the major capsid protein, in cell-free lysates by protein mass spectrometry. Comparative proteomics of protein extracts of infected E. coli cells revealed that proteins involved in amino acid and protein metabolism were more abundant in infected compared to non-infected cells. Among the proteins involved in the stress response, 74% were less abundant in the infected cultures compared to the non-infected controls, with six proteins showing significant less abundance. Repressing the expression of these proteins may be a phage strategy to evade host defense mechanisms. Our results contribute to diversifying phage collections, identifying structural proteins to enable better reliability in annotating taxonomically related phage genomes, and understanding phage-host interactions at the protein level.

Phage Genome Diversity in a Biogas-Producing Microbiome Analyzed by Illumina and Nanopore GridION Sequencing.

The microbial biogas network is complex and intertwined, and therefore relatively stable in its overall functionality. However, if key functional groups of microorganisms are affected by biotic or abiotic factors, the entire efficacy may be impaired. Bacteriophages are hypothesized to alter the steering process of the microbial network. In this study, an enriched fraction of virus-like particles was extracted from a mesophilic biogas reactor and sequenced on the Illumina MiSeq and Nanopore GridION sequencing platforms. Metagenome data analysis resulted in identifying 375 metagenome-assembled viral genomes (MAVGs). Two-thirds of the classified sequences were only assigned to the superkingdom Viruses and the remaining third to the family Siphoviridae, followed by Myoviridae, Podoviridae, Tectiviridae, and Inoviridae. The metavirome showed a close relationship to the phage genomes that infect members of the classes Clostridia and Bacilli. Using publicly available biogas metagenomic data, a fragment recruitment approach showed the widespread distribution of the MAVGs studied in other biogas microbiomes. In particular, phage sequences from mesophilic microbiomes were highly similar to the phage sequences of this study. Accordingly, the virus particle enrichment approach and metavirome sequencing provided additional genome sequence information for novel virome members, thus expanding the current knowledge of viral genetic diversity in biogas reactors.

Biosignature stability in space enables their use for life detection on Mars.

Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments, Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable biomolecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.

Who put the film in biofilm? The migration of a term from wastewater engineering to medicine and beyond.

Sessile microorganisms were described as early as the seventeenth century. However, the term biofilm arose only in the 1960s in wastewater treatment research and was adopted later in marine fouling and in medical and dental microbiology. The sessile mode of microbial life was gradually recognized to be predominant on Earth, and the term biofilm became established for the growth of microorganisms in aggregates, frequently associated with interfaces, although many, if not the majority, of them not being continuous "films" in the strict sense. In this sessile form of life, microorganisms live in close proximity in a matrix of extracellular polymeric substances (EPS). They share emerging properties, clearly distinct from solitary free floating planktonic microbial cells. Common characteristics include the formation of synergistic microconsortia, using the EPS matrix as an external digestion system, the formation of gradients and high biodiversity over microscopically small distances, resource capture and retention, facilitated gene exchange as well as intercellular communication, and enhanced tolerance to antimicrobials. Thus, biofilms belong to the class of collective systems in biology, like forests, beehives, or coral reefs, although the term film addresses only one form of the various manifestations of microbial aggregates. The uncertainty of this term is discussed, and it is acknowledged that it will not likely be replaced soon, but it is recommended to understand these communities in the broader sense of microbial aggregates.

Effect of a Profound Feedstock Change on the Structure and Performance of Biogas Microbiomes.

In this study the response of biogas-producing microbiomes to a profound feedstock change was investigated. The microbiomes were adapted to the digestion of either 100% sugar beet, maize silage, or of the silages with elevated amounts of total ammonium nitrogen (TAN) by adding ammonium carbonate or animal manure. The feedstock exchange resulted in a short-range decrease or increase in the biogas yields according to the level of chemical feedstock complexity. Fifteen taxa were found in all reactors and can be considered as generalists. Thirteen taxa were detected in the reactors operated with low TAN and six in the reactors with high TAN concentration. Taxa assigned to the phylum Bacteroidetes and to the order Spirochaetales increased with the exchange to sugar beet silage, indicating an affinity to easily degradable compounds. The recorded TAN-sensitive taxa (phylum Cloacimonetes) showed no specific affinity to maize or sugar beet silage. The archaeal community remained unchanged. The reported findings showed a smooth adaptation of the microbial communities, without a profound negative impact on the overall biogas production indicating that the two feedstocks, sugar beet and maize silage, potentially do not contain chemical compounds that are difficult to handle during anaerobic digestion.

Manganese-oxidizing bacteria isolated from natural and technical systems remove cylindrospermopsin.

The cyanotoxin cylindrospermopsin was discovered during a drinking water-related outbreak of human poisoning in 1979. Knowledge about the degradation of cylindrospermopsin in waterbodies is limited. So far, only few cylindrospermopsin-removing bacteria have been described. Manganese-oxidizing bacteria remove a variety of organic compounds. However, this has not been assessed for cyanotoxins yet. We investigated cylindrospermopsin removal by manganese-oxidizing bacteria, isolated from natural and technical systems. Cylindrospermopsin removal was evaluated under different conditions. We analysed the correlation between the amount of oxidized manganese and the cylindrospermopsin removal, as well as the removal of cylindrospermopsin by sterile biogenic oxides. Removal rates in the range of 0.4-37.0 µg L-1 day-1 were observed. When MnCO3 was in the media Pseudomonas sp. OF001 removed ∼100% of cylindrospermopsin in 3 days, Comamonadaceae bacterium A210 removed ∼100% within 14 days, and Ideonella sp. A288 and A226 removed 65% and 80% within 28 days, respectively. In the absence of Mn2+, strain A288 did not remove cylindrospermopsin, while the other strains removed 5-16%. The amount of manganese oxidized by the strains during the experiment did not correlate with the amount of cylindrospermopsin removed. However, the mere oxidation of Mn2+ was indispensable for cylindrospermopsin removal. Cylindrospermopsin removal ranging from 0 to 24% by sterile biogenic oxides was observed. Considering the efficient removal of cylindrospermopsin by the tested strains, manganese-oxidizing bacteria might play an important role in cylindrospermopsin removal in the environment. Besides, manganese-oxidizing bacteria could be promising candidates for biotechnological applications for cylindrospermopsin removal in water treatment plants.

Manganese-oxidizing bacteria form multiple cylindrospermopsin transformation products with reduced human liver cell toxicity.

Cylindrospermopsin (CYN) is a toxic alkaloid highly persistent in aquatic environments. Biological removal of CYN was described previously. However, no transformation products formed by biological processes could be identified so far. Here, we describe that various manganese-oxidizing bacteria (MOB) transform CYN completely at an initial mean concentration of 7 mg L-1 (17 µM) within 3 to 34 days. Regardless of the strain, and transformation rate, transformation of CYN by MOB led to the same seven transformation products identified by mass spectrometry, which suggests that the removal of CYN by MOB follows a similar mechanism. Oxidation was the main transformation process, and the uracil moiety was the most susceptible part of the CYN molecule. In vitro cytotoxicity tests with the transformation products of CYN formed by one of the tested strains against the two human liver cell lines HepG2 and HepaRG, revealed that the transformation products were substantially less toxic than pure CYN for both cell lines. The results suggest that incubation with MOB might be an option for water treatment to remove CYN and may allow more detailed studies on the fate of CYN in the environment.

Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European artificial groundwater recharge systems.

Aquifer recharge presents advantages for integrated water management in the anthropic cycle, namely, advanced treatment of reclaimed water and additional dilution of pollutants due to mixing with natural groundwater. Nevertheless, this practice represents a health and environmental hazard because of the presence of pathogenic microorganisms and chemical contaminants. To assess the quality of water extracted from recharged aquifers, the groundwater recharge systems in Torreele, Belgium, Sabadell, Spain, and Nardò, Italy, were investigated for fecal-contamination indicators, bacterial pathogens, and antibiotic resistance genes over the period of 1 year. Real-time quantitative PCR assays for Helicobacter pylori, Yersinia enterocolitica, and Mycobacterium avium subsp. paratuberculosis, human pathogens with long-time survival capacity in water, and for the resistance genes ermB, mecA, blaSHV-5, ampC, tetO, and vanA were adapted or developed for water samples differing in pollutant content. The resistance genes and pathogen concentrations were determined at five or six sampling points for each recharge system. In drinking and irrigation water, none of the pathogens were detected. tetO and ermB were found frequently in reclaimed water from Sabadell and Nardò. mecA was detected only once in reclaimed water from Sabadell. The three aquifer recharge systems demonstrated different capacities for removal of fecal contaminators and antibiotic resistance genes. Ultrafiltration and reverse osmosis in the Torreele plant proved to be very efficient barriers for the elimination of both contaminant types, whereas aquifer passage followed by UV treatment and chlorination at Sabadell and the fractured and permeable aquifer at Nardò posed only partial barriers for bacterial contaminants.

Interactions between hyphosphere-associated bacteria and the fungus Cladosporium herbarum on aquatic leaf litter.

We investigated microbial interactions of aquatic bacteria associated with hyphae (the hyphosphere) of freshwater fungi on leaf litter. Bacteria were isolated directly from the hyphae of fungi from sedimented leaves of a small stream in the National Park "Lower Oder," Germany. To investigate interactions, bacteria and fungi were pairwise co-cultivated on leaf-extract medium and in microcosms loaded with leaves. The performance of fungi and bacteria was monitored by measuring growth, enzyme production, and respiration of mono- and co-cultures. Growth inhibition of the fungus Cladosporium herbarum by Ralstonia pickettii was detected on leaf extract agar plates. In microcosms, the presence of Chryseobacterium sp. lowered the exocellulase, endocellulase, and cellobiase activity of the fungus. Additionally, the conversion of leaf material into microbial biomass was retarded in co-cultures. The respiration of the fungus was uninfluenced by the presence of the bacterium.

Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS.

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports-among others-the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit.

In situ detection of freshwater fungi in an alpine stream by new taxon-specific fluorescence in situ hybridization probes.

New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes.

Complexity of ultrafiltration membrane fouling caused by macromolecular dissolved organic compounds in secondary effluents.

Recent investigations indicate the relevance of extracellular polymeric substances (EPS) in terms of fouling of low-pressure membranes in advanced wastewater treatment. In this study, the high impact of the macromolecular fraction of effluent organic matter on fouling was confirmed in cross-flow ultrafiltration experiments using secondary effluent with and without autochthonous biopolymers. A method for the extraction of a natural mixture of EPS derived from the bacterium Sinorhizobium sp. is presented. Ultrafiltration of solutions of this bacterial EPS extract revealed a correlation between the concentration of EPS and the loss of permeate flux. However, in ultrafiltration tests using extracted bacterial EPS in a model solution as well as in secondary effluent without autochthonous biopolymers, the extent of membrane fouling was not identical with the fouling provoked by secondary effluent organic matter, although the biopolymer concentrations were comparable. The differences in the fouling behaviour of the extracted bacterial EPS and effluent organic matter are considered to be due to different compositions of the biopolymer fraction in terms of proteins, polysaccharides, and other organic colloids, indicating a particular impact of proteins on ultrafiltration membrane fouling.

Environmentally relevant concentrations of pharmaceuticals influence the initial adhesion of bacteria.

Pharmaceuticals are abundant at low concentrations (i.e. ng L(-1); microg L(-1)) in natural aquatic systems. However, very little is known about their effects on microorganisms. This study investigated the influence of the pharmaceuticals phenazone, amoxicillin and erythromycin, at low, non-toxic concentrations (i.e. 0.5-50 microg L(-1)), on the initial adhesion of bacteria to uncoated and iron-coated polystyrene. The influence of the pharmaceuticals on the initial adhesion of bacterial pure cultures (Escherichia coli, Aquabacterium commune, Bacillus subtilis) isolated from natural aquatic systems, was investigated with a plate assay. Initial adhesion of the pure cultures depended on the selected pharmaceutical, its concentration, the bacterial strain and the adhesion surface. Different combinations of these parameters resulted in inhibition, enhancement or had no effect on initial adhesion. In addition, a continuous flow system was used to investigate the influence of the pharmaceuticals on the initial adhesion of a drinking water microbial community. The drinking water community showed decreased adhesion in the presence of the pharmaceuticals regardless of adhesion surface. The results show that pharmaceuticals at environmentally relevant concentrations can influence the initial adhesion of bacteria. Thus, pharmaceutical compounds that are introduced to natural aquatic systems are able to exert subtle effects on bacteria.

Draft Genome Sequence of Strain B 225, an Iron-Depositing Isolate of the Genus Novosphingobium.

Here, we report the draft genome sequence of Novosphingobium sp. strain B 225, an iron-depositing bacterium isolated from a phenazone-amended naturally grown biofilm. This biofilm was grown in the Unteres Odertal National Park, Germany. Illumina NextSeq sequencing was used to determine the genome of the strain.

Draft Genome Sequence of Actinobacterial Strain Kineosporia sp. R_H_3, a Neutrophilic Iron-Depositing Bacterium.

The draft genome sequence of a neutrophilic iron-depositing actinobacterial strain, Kineosporia sp. R_H_3, is reported here. Detailed analysis of the genome can elucidate the role of specific cytochromes for Fe oxidation and how this organism might receive energy from Fe oxidation. To date, this is the second publicly available genome sequence of a Kineosporia strain.

Opening the black box of spring water microbiology from alpine karst aquifers to support proactive drinking water resource management.

Over the past 15 years, pioneering interdisciplinary research has been performed on the microbiology of hydrogeologically well-defined alpine karst springs located in the Northern Calcareous Alps (NCA) of Austria. This article gives an overview on these activities and links them to other relevant research. Results from the NCA springs and comparable sites revealed that spring water harbors abundant natural microbial communities even in aquifers with high water residence times and the absence of immediate surface influence. Apparently, hydrogeology has a strong impact on the concentration and size of the observed microbes, and total cell counts (TCC) were suggested as a useful means for spring type classification. Measurement of microbial activities at the NCA springs revealed extremely low microbial growth rates in the base flow component of the studied spring waters and indicated the importance of biofilm-associated microbial activities in sediments and on rock surfaces. Based on genetic analysis, the autochthonous microbial endokarst community (AMEC) versus transient microbial endokarst community (TMEC) concept was proposed for the NCA springs, and further details within this overview article are given to prompt its future evaluation. In this regard, it is well known that during high-discharge situations, surface-associated microbes and nutrients such as from soil habitats or human settlements-potentially containing fecal-associated pathogens as the most critical water-quality hazard-may be rapidly flushed into vulnerable karst aquifers. In this context, a framework for the comprehensive analysis of microbial pollution has been proposed for the NCA springs to support the sustainable management of drinking water safety in accordance with recent World Health Organization guidelines. Near-real-time online water quality monitoring, microbial source tracking (MST) and MST-guided quantitative microbial-risk assessment (QMRA) are examples of the proposed analytical tools. In this context, this overview article also provides a short introduction to recently emerging methodologies in microbiological diagnostics to support reading for the practitioner. Finally, the article highlights future research and development needs. This article is categorized under: 1Engineering Water > Water, Health, and Sanitation2Science of Water > Water Extremes3Water and Life > Nature of Freshwater Ecosystems.

Draft Genome Sequence of the Gram-Positive Neutrophilic Iron-Precipitating Kineosporia sp. Strain A_224.

We report here the draft genome sequence of the neutrophilic iron-precipitating Kineosporia sp. strain A_224. Analysis of the predicted genes may improve our knowledge of its role in ochrous formations in natural and technical water systems. This is the first public genome sequence of a Kineosporia aurantiaca strain.