Scleromatobacter humisilvae gen. nov., sp. nov., a novel bacterium isolated from oak forest soil.

A novel bacterial strain, designated BS-T2-15T, isolated from forest soil in close proximity to decaying oak wood, was characterized using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences as well as phylogenomic analyses based on coding sequences of 340 concatenated core proteins indicated that strain BS-T2-15T forms a distinct and robust lineage in the Rubrivivax-Roseateles -Leptothrix-Azohydromonas -Aquincola-Ideonella branch of the order Burkholderiales. The amino acid identity and the percentage of conserved proteins between the genome of strain BS-T2-15T and genomes of closely related type strains ranged from 64.27 to 66.57% and from 40.89 to 49.27 %, respectively, providing genomic evidence that strain BS-T2-15T represents a new genus. Its cells are Gram-stain-negative, aerobic, motile by a polar flagellum, rod-shaped and form incrusted white to ivory colonies. Optimal growth is observed at 20-22 °C, pH 6 and 0% NaCl. The predominant fatty acids of strain BS-T2-15T are C16 : 1 ω7c, C16 : 0 and C14 : 0 2-OH. Its polar lipid profile consists of a mixture of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol and its main respiratory quinone is ubiquinone 8. The estimated size of its genome is 6.28 Mb with a DNA G+C content of 69.56 mol%. Therefore, on the basis of phenotypic and genotypic properties, the new strain BS-T2-15T represents a novel genus and species for which the name Scleromatobacter humisilvae gen. nov., sp. nov., is proposed. The type strain is BS-T2-15T (DSM 113115T=UBOCC-M-3373T).

Acidisoma silvae sp. nov. and Acidisomacellulosilytica sp. nov., Two Acidophilic Bacteria Isolated from Decaying Wood, Hydrolyzing Cellulose and Producing Poly-3-hydroxybutyrate.

Two novel strains, HW T2.11T and HW T5.17T, were isolated from decaying wood (forest of Champenoux, France). Study of the 16S rRNA sequence similarity indicated that the novel strains belong to the genus Acidisoma. The sequence similarity of the 16S rRNA gene of HW T2.11T with the corresponding sequences of A. tundrae and A. sibiricum was 97.30% and 97.25%, while for HW T5.17T it was 96.85% and 97.14%, respectively. The DNA G+C contents of the strains were 62.32-62.50%. Cells were Gram-negative coccobacilli that had intracellular storage granules (poly-3-hydroxybutyrate (P3HB)) that confer resistance to environmental stress conditions. They were mesophilic and acidophilic organisms growing at 8-25 °C, at a pH of 2.0-6.5, and were capable of using a wide range of organic compounds and complex biopolymers such as starch, fucoidan, laminarin, pectin and cellulose, the latter two being involved in wood composition. The major cellular fatty acid was cyclo C19:0ω8c and the major quinone was Q-10. Overall, genome relatedness indices between genomes of strains HW T2.11T and HW T5.17T (Orthologous Average Nucleotide Identity (OrthoANI) value = 83.73% and digital DNA-DNA hybridization score = 27.5%) confirmed that they belonged to two different species. Genetic predictions indicate that the cyclopropane fatty acid (CFA) pathway is present, conferring acid-resistance properties to the cells. The two novel strains might possess a class IV polyhydroxyalcanoate (PHA) synthase operon involved in the P3HB production pathway. Overall, the polyphasic taxonomic analysis shows that these two novel strains are adapted to harsh environments such as decaying wood where the organic matter is difficult to access, and can contribute to the degradation of dead wood. These strains represent novel species of the genus Acidisoma, for which the names Acidisoma silvae sp. nov. and Acidisomacellulosilytica sp. nov. are proposed. The type strains of Acidisoma silvae and Acidisomacellulosilytica are, respectively, HW T2.11T (DSM 111006T; UBOCC-M-3364T) and HW T5.17T (DSM 111007T; UBOCC-M-3365T).

Sapwood and heartwood affect differentially bacterial and fungal community structure and successional dynamics during Quercus petraea decomposition.

In forests, bacteria and fungi are key players in wood degradation. Still, studies focusing on bacterial and fungal successions during the decomposition process depending on the wood types (i.e. sapwood and heartwood) remain scarce. This study aimed to understand the effect of wood type on the dynamics of microbial ecological guilds in wood decomposition. Using Illumina metabarcoding, bacterial and fungal communities were monitored every 3 months for 3 years from Quercus petraea wood discs placed on forest soil. Wood density and microbial enzymes involved in biopolymer degradation were measured. We observed rapid changes in the bacterial and fungal communities and microbial ecological guilds associated with wood decomposition throughout the experiment. Bacterial and fungal succession dynamics were very contrasted between sapwood and heartwood. The initial microbial communities were quickly replaced by new bacterial and fungal assemblages in the sapwood. Conversely, some initial functional guilds (i.e. endophytes and yeasts) persisted all along the experiment in heartwood and finally became dominant, possibly limiting the development of saprotrophic fungi. Our data also suggested a significant role of bacteria in nitrogen cycle during wood decomposition.

Bacterial-fungal interactions: ecology, mechanisms and challenges.

Fungi and bacteria are found living together in a wide variety of environments. Their interactions are significant drivers of many ecosystem functions and are important for the health of plants and animals. A large number of fungal and bacterial families engage in complex interactions that lead to critical behavioural shifts of the microorganisms ranging from mutualism to antagonism. The importance of bacterial-fungal interactions (BFI) in environmental science, medicine and biotechnology has led to the emergence of a dynamic and multidisciplinary research field that combines highly diverse approaches including molecular biology, genomics, geochemistry, chemical and microbial ecology, biophysics and ecological modelling. In this review, we discuss recent advances that underscore the roles of BFI across relevant habitats and ecosystems. A particular focus is placed on the understanding of BFI within complex microbial communities and in regard of the metaorganism concept. We also discuss recent discoveries that clarify the (molecular) mechanisms involved in bacterial-fungal relationships, and the contribution of new technologies to decipher generic principles of BFI in terms of physical associations and molecular dialogues. Finally, we discuss future directions for research in order to stimulate synergy within the BFI research area and to resolve outstanding questions.

Topographic cues guide the attachment of diatom cells and algal zoospores.

Surface topography plays a key role in the colonization of substrata by the colonizing stages of marine fouling organisms. For the innovation of marine antifouling coatings, it is essential to understand how topographic cues affect the settlement of these organisms. In this study, tapered, spiked microstructures and discrete honeycombs of varying feature dimensions were designed and fabricated in order to examine the influence of topography on the attachment of zoospores of the green macroalga Ulva linza and cells of the diatom (microalga) Navicula incerta. Contrasting results were obtained with these two species of algae. Indeed, the preferred location of cells of N. incerta was dominated by attachment point theory, which suggested a positive correlation between the density of cells adhering and the amount of available attachment points, while the settlement of spores of U. linza was mainly regulated by both Wenzel roughness and local binding geometry.

Model Amphiphilic Block Copolymers with Tailored Molecular Weight and Composition in PDMS-Based Films to Limit Soft Biofouling.

A set of controlled surface composition films was produced utilizing amphiphilic block copolymers dispersed in a cross-linked poly(dimethylsiloxane) network. These block copolymers contained oligo(ethylene glycol) (PEGMA) and fluoroalkyl (AF6) side chains in selected ratios and molecular weights to control surface chemistry including antifouling and fouling-release performance. Such properties were assessed by carrying out assays using two algae, the green macroalga Ulva linza (favors attachment to polar surfaces) and the unicellular diatom Navicula incerta (favors attachment to nonpolar surfaces). All films performed well against U. linza and exhibited high removal of attached sporelings (young plants) under an applied shear stress, with the lower molecular weight block copolymers being the best performing in the set. The composition ratios from 50:50 to 60:40 of the AF6/PEGMA side groups were shown to be more effective, with several films exhibiting spontaneous removal of the sporelings. The cells of N. incerta were also removed from several coating compositions. All films were characterized by surface techniques including captive bubble contact angle, atomic force microscopy, and near edge X-ray absorption fine structure spectroscopy to correlate surface chemistry and morphology with biological performance.

Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk.

The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensis K.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifying their organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the action mechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability, membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3days incubation in control supernatant, while it was prevented for up to 10days by the active supernatant. Interestingly, between 66 and 99% of yeast cells were under a viable but non-cultivable (VNC) state despite an absence of membrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.

Antialgal activity of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes against the marine alga Ulva.

Marine biofouling has detrimental effects on the environment and economy, and current antifouling coatings research is aimed at environmentally benign, non-toxic materials. The possibility of using contact-active coatings is explored, by considering the antialgal activity of cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes. The antialgal activity was investigated via zoospore settlement and sporeling growth assays of the marine algae Ulva linza and U. lactuca. The assay results for PDMAEMA brushes were compared to those for anionic and neutral surfaces. It was found that only PDMAEMA could disrupt zoospores that come into contact with it, and that it also inhibits the subsequent growth of normally settled spores. Based on the spore membrane properties, and characterization of the PDMAEMA brushes over a wide pH range, it is hypothesized that the algicidal mechanisms are similar to the bactericidal mechanisms of cationic polymers, and that further development could lead to successful contact-active antialgal coatings.

Charged hydrophilic polymer brushes and their relevance for understanding marine biofouling.

The resistance of charged polymers to biofouling was investigated by subjecting cationic (PDMAEMA), anionic (PSPMA), neutral (PHEMA-co-PEG10MA), and zwitterionic (PSBMA) brushes to assays testing protein adsorption; attachment of the marine bacterium Cobetia marina; settlement and adhesion strength of zoospores of the green alga Ulva linza; settlement of barnacle (Balanus amphitrite and B. improvisus) cypris larvae; and field immersion tests. Several results go beyond the expected dependence on direct electrostatic attraction; PSPMA showed good resistance towards attachment of C. marina, low settlement and adhesion of U. linza zoospores, and significantly lower biofouling than on PHEMA-co-PEG10MA or PSBMA after a field test for one week. PDMAEMA showed potential as a contact-active anti-algal coating due to its capacity to damage attached spores. However, after field testing for eight weeks, there were no significant differences in biofouling coverage among the surfaces. While charged polymers are unsuitable as antifouling coatings in the natural environment, they provide valuable insights into fouling processes, and are relevant for studies due to charging of nominally neutral surfaces.

Hydration and chain entanglement determines the optimum thickness of poly(HEMA-co-PEG10MA) brushes for effective resistance to settlement and adhesion of marine fouling organisms.

Understanding how surface physicochemical properties influence the settlement and adhesion of marine fouling organisms is important for the development of effective and environmentally benign marine antifouling coatings. We demonstrate that the thickness of random poly(HEMA-co-PEG10MA) copolymer brushes affect antifouling behavior. Films of thicknesses ranging from 50 to 1000 Å were prepared via surface-initiated atom-transfer radical polymerization and characterized using infrared spectroscopy, ellipsometry, atomic force microscopy and contact angle measurements. The fouling resistance of these films was investigated by protein adsorption, attachment of the marine bacterium Cobetia marina, settlement and strength of attachment tests of zoospores of the marine alga Ulva linza and static immersion field tests. These assays show that the polymer film thickness influenced the antifouling performance, in that there is an optimum thickness range, 200-400 Å (dry thickness), where fouling of all types, as well as algal spore adhesion, was lower. Field test results also showed lower fouling within the same thickness range after 2 weeks of immersion. Studies by quartz crystal microbalance with dissipation and underwater captive bubble contact angle measurements show a strong correlation between lower fouling and higher hydration, viscosity and surface energy of the poly(HEMA-co-PEG10MA) brushes at thicknesses around 200-400 Å. We hypothesize that the reduced antifouling performance is caused by a lower hydration capacity of the polymer for thinner films, and that entanglement and crowding in the film reduces the conformational freedom, hydration capacity and fouling resistance for thicker films.

Interactions between microbial biofilms and marine fouling algae: a mini review.

Natural and artificial substrata immersed in the marine environment are typically colonized by microorganisms, which may moderate the settlement/recruitment of algal spores and invertebrate larvae of macrofouling organisms. This mini-review summarizes the major interactions occurring between microbial biofilms and marine fouling algae, including their effects on the settlement, growth and morphology of the adult plants. The roles of chemical compounds that are produced by both bacteria and algae and which drive the interactions are reviewed. The possibility of using such bioactive compounds to control macrofouling will be discussed.

Slippery liquid-infused porous surfaces showing marine antibiofouling properties.

Marine biofouling is a longstanding problem because of the constant challenges placed by various fouling species and increasingly restricted environmental regulations for antifouling coatings. Novel nonbiocidal strategies to control biofouling will necessitate a multifunctional approach to coating design. Here we show that slippery liquid-infused porous surfaces (SLIPSs) provide another possible strategy to obtaining promising antifouling coatings. Microporous butyl methacrylate-ethylene dimethacrylate (BMA-EDMA) surfaces are prepared via UV-initiated free-radical polymerization. Subsequent infusion of fluorocarbon lubricants (Krytox103, Krytox100, and Fluorinert FC-70) into the porous microtexture results in liquid-repellent slippery surfaces. To study the interaction with marine fouling organisms, settlement of zoospores of the alga Ulva linza and cypris larvae of the barnacle Balanus amphitrite is tested in laboratory assays. BMA-EDMA surfaces infused with Krytox103 and Krytox100 exhibit remarkable inhibition of settlement (attachment) of both spores and cyprids to a level comparable to that of a poly(ethylene glycol) (PEG)-terminated self-assembled monolayer. In addition, the adhesion strength of sporelings (young plants) of U. linza is reduced for BMA-EDMA surfaces infused with Krytox103 and Krytox100 compared to pristine (noninfused) BMA-EDMA and BMA-EDMA infused with Fluorinert FC-70. Immersion tests suggest a correlation between the stability of slippery coatings in artificial seawater and fouling resistance efficacy. The results indicate great potential for the application of this concept in fouling-resistant marine coatings.

Photochemically prepared, two-component polymer-concentration gradients.

A versatile, photochemical surface-modification approach using nitrene-insertion reactions has been employed to develop an ultrathin, two-component, polymer-gradient coating. Perfluorophenyl azide (PFPA) acted as the photosensitive moiety, forming a nitrene radical upon 254 nm UV exposure. Cationic poly(allyl amine) was grafted with PFPA and surface-anchored onto silicon wafers by means of electrostatic self-assembly. After spin-coating of polystyrene (PS), the substrate was illuminated from behind a moving shutter, thereby controlling the azide-to-nitrene conversion degree across the substrate, and leading to a gradually varying PS density after rinsing. Backfilling with poly(vinyl pyrrolidone) (PVP) and re-exposing to UV light formed a two-component polymer-density gradient. The composition varied linearly following exposure to a linear UV exposure profile, as determined with spectroscopic ellipsometry (ELM) and X-ray photoelectron spectroscopy (XPS). High-spatial-resolution, time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed a high degree of mixing between the two incompatible polymers on the micrometer scale. The dynamic water-contact angle (dCA) was found to depend strongly on the sample history, suggesting adaptive properties of the coating, which was further confirmed by angle-resolved XPS (ARXPS). To confirm the applicability of the system for biological investigations, gradients were exposed to zoospores of the macrofouling alga Ulva linza , and a critical PS composition of 70% was identified, above which settlement started to increase. It has been shown that a two-component polymer-density gradient can provide a high-throughput platform for determining critical surface properties of polymer blend materials.

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources.

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation.

An electrically reversible switchable surface to control and study early bacterial adhesion dynamics in real-time.

Bacterial adhesion can be controlled by applying electrical potentials to surfaces incorporating well-spaced negatively charged 11-mercaptoundecanoic acids. When combined with electrochemical surface plasmon resonance, these dynamic surfaces become powerful for monitoring and analysing the passage between reversible and non-reversible cell adhesion, opening new opportunities to advance our understanding of cell adhesion processes.

Effect of bacterial biofilms formed on fouling-release coatings from natural seawater and Cobetia marina, on the adhesion of two marine algae.

Previous studies have shown that bacterial biofilms formed from natural seawater (NSW) enhance the settlement of spores of the green alga Ulva linza, while single-species biofilms may enhance or reduce settlement, or have no effect at all. However, the effect of biofilms on the adhesion strength of algae, and how that may be influenced by coating/surface properties, is not known. In this study, the effect of biofilms formed from natural seawater and the marine bacterium Cobetia marina, on the settlement and the adhesion strength of spores and sporelings of the macroalga U. linza and the diatom Navicula incerta, was evaluated on Intersleek(®) 700, Intersleek(®) 900, poly(dimethylsiloxane) and glass. The settlement and adhesion strength of these algae were strongly influenced by biofilms and their nature. Biofilms formed from NSW enhanced the settlement (attachment) of both algae on all the surfaces while the effect of biofilms formed from C. marina varied with the coating type. The adhesion strength of spores and sporelings of U. linza and diatoms was reduced on all the surfaces biofilmed with C. marina, while adhesion strength on biofilms formed from NSW was dependent on the alga (and on its stage of development in the case of U. linza), and coating type. The results illustrate the complexity of the relationships between fouling algae and bacterial biofilms and suggest the need for caution to avoid over-generalisation.

Microbial and chemical markers: runoff transfer in animal manure-amended soils.

Fecal contamination of water resources is evaluated by the enumeration of the fecal coliforms and Enterococci. However, the enumeration of these indicators does not allow us to differentiate between the sources of fecal contamination. Therefore, it is important to use alternative indicators of fecal contamination to identify livestock contamination in surface waters. The concentration of fecal indicators (, enteroccoci, and F-specific bacteriophages), microbiological markers (Rum-2-bac, Pig-2-bac, and ), and chemical fingerprints (sterols and stanols and other chemical compounds analyzed by 3D-fluorescence excitation-matrix spectroscopy) were determined in runoff waters generated by an artificial rainfall simulator. Three replicate plot experiments were conducted with swine slurry and cattle manure at agronomic nitrogen application rates. Low amounts of bacterial indicators (1.9-4.7%) are released in runoff water from swine-slurry-amended soils, whereas greater amounts (1.1-28.3%) of these indicators are released in runoff water from cattle-manure-amended soils. Microbial and chemical markers from animal manure were transferred to runoff water, allowing discrimination between swine and cattle fecal contamination in the environment via runoff after manure spreading. Host-specific bacterial and chemical markers were quantified for the first time in runoff waters samples after the experimental spreading of swine slurry or cattle manure.

Estimation of pig fecal contamination in a river catchment by real-time PCR using two pig-specific Bacteroidales 16S rRNA genetic markers.

The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two pig markers, 98 to 100% sensitivity and 100% specificity were obtained when tested by TaqMan real-time PCR. A decrease in the concentrations of Pig-1-Bac and Pig-2-Bac markers was observed throughout the slurry treatment chain. The two newly designed pig-specific Bacteroidales markers, plus the human-specific (HF183) and ruminant-specific (BacR) Bacteroidales markers, were then applied to river water samples (n = 24) representing 14 different sites from the French Daoulas River catchment (Brittany, France). Pig-1-Bac and Pig-2-Bac were quantified in 25% and 62.5%, respectively, of samples collected around pig farms, with concentrations ranging from 3.6 to 4.1 log10 copies per 100 ml of water. They were detected in water samples collected downstream from pig farms but never detected near cattle farms. HF183 was quantified in 90% of water samples collected downstream near Daoulas town, with concentrations ranging between 3.6 and 4.4 log10 copies per 100 ml of water, and BacR in all water samples collected around cattle farms, with concentrations ranging between 4.6 and 6.0 log10 copies per 100 ml of water. The results of this study highlight that pig fecal contamination was not as frequent as human or bovine fecal contamination and that fecal pollution generally came from multiple origins. The two pig-specific Bacteroidales markers can be applied to environmental water samples to detect pig fecal pollution.