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Ultraviolet-C light-emitting diodes (UV-C LEDs) are an emerging technology for decontamination applications in different sectors. In this study, the inactivation of bacterial biofilms was investigated by applying an UV-C LED emitting at 280 nm and by measuring both the influence of the initial cell density (load) and presence of an extracellular matrix (biofilm). Two bacterial strains exposing diverging matrix structures and biochemical compositions were used: Pseudomonas aeruginosa and Leuconostoc citreum. UV-C LED irradiation was applied at three UV doses (171 to 684 mJ/cm 2 ) on both surface-spread cells and on 24-h biofilms and under controlled cell loads, and bacterial survival was determined. All surface-spread bacteria, between 10 5 and 10 9  CFU/cm 2 , and biofilms at 10 8  CFU/cm 2 showed that bacterial response to irradiation was dose-dependent. The treatment efficacy decreased significantly for L. citreum surface-spread cells when the initial cell load was high, while no load effect was observed for P. aeruginosa. Inactivation was also reduced when bacteria were grown under a biofilm form, especially for P. aeruginosa: a protective effect could be attributed to abundant extracellular DNA and proteins in the matrix of P. aeruginosa biofilms, as revealed by Confocal Laser Scanning Microscopy observations. This study showed that initial cell load and exopolymeric substances are major factors influencing UV-C LED antibiofilm treatment efficacy. KEY POINTS: • Bacterial cell load (CFU/cm 2 ) could impact UV-C LED irradiation efficiency • Characteristics of the biofilm matrix have a paramount importance on inactivation • The dose to be applied can be predicted based on biofilm properties., (© 2024. The Author(s).)

The valorization of biological aggregates through the extraction of hydrogel-forming polymers can enhance the economics and sustainability of various processes in which bacteria are involved in organic waste transformation, such as wastewater treatment. Achieving these goals requires the development of a method capable of detecting the presence of gel-forming polymers in complex mixtures containing biopolymers that are most often unknown and uncharacterized. A miniaturized screening method capable of detecting gelation via ionic crosslinking using only 1 to 3 mg of the tested samples (commercial molecules or extracellular polymeric substances, EPSs) is proposed. The method consists of calculating a percentage of reactivity (%R) through UV-vis spectra and determining the percentage of gel volume (%Vg) formed after the addition of calcium. Both factors were combined to give a gelling factor (GF), and the test was applied to pure commercial molecules (BSA, DNA, alginate (ALV), and a mixture of them), allowing the classification of the following solutions according to their gel-forming capacity: GF (ALV) > GF (ALV+DNA) > GF (BSA+ALV+DNA) > GF (BSA+ALV) > GF (DNA) > GF (BSA+DNA) > GF (BSA) . As a relevant tool for screening hydrogel-forming solutions, the method was applied to the EPS extracted from aerobic granular sludge. The EPS (0.5% w / v ) had a GF of 0.16 ± 0.03, equivalent to approximately half of the GF of ALV (0.38 ± 0.02 at 0.5% w / v ). The developed test pushes the limits of the existing gel-detection techniques because it allows for quicker, less consuming, and more informative gelation detection through the use of simple methods that do not require sophisticated equipment.

Bacillus amyloliquefaciens is a ubiquitous soil and plant-associated bacterial species which shows structural and adaptative responses to the environment. This present paper explores the ability of the strain L-17 to form subaerial biofilms on a liquid surface. Hydrophobic and non-wetting properties were observed for the rough top biofilm layer in contact with the air, which are quite different to the hydrophilic properties which were observed for the smooth biofilm layer in contact with the liquid. Both pellicle interfaces were visualized by scanning electron microscopy revealing a complex three-dimensional architecture composed of exopolymers organized in stacked fibrous network or sheet-like structures in the vicinity of the subaerial surface. Disruption of the extracellular matrix by combining physical and chemical treatments indicated that both loosely and tightly bound polysaccharides were found as major components of this complex pellicle. Proteins were also involved in the aggregation and cohesion of the matrix as multi extraction steps were needed to recover some tightly bounded proteins. This was confirmed by applying protease treatment which was able to significantly disrupt the pellicle. Overall results underline the ability of B. amyloliquefaciens L-17 to survive on air-liquid interfaces. This feature offers an interesting strategy to escape aquatic environments and develop aerial biofilm in response to environmental changes involving wet-dry cycles., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Bacillus amyloliquefaciens L-17 strain was isolated from a sample of chicken feathers. Here, we report complete genome sequence data of B. amyloliquefaciens L-17. The size of the genome is 3,933,788 bp which harbours 4001 coding Sequences. The BioProject has been deposited at NCBI GenBank. The GenBank accession numbers are PRJNA727793 for the BioProject, CP074391.1 for the chromosome, GCA_018363035.1 for GenBank assembly accession and SAMN19035411 for the BioSample., Competing Interests: The authors declare that they do not have conflict of interest that could influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier Inc.)

Phototrophic biofilms are exposed to multiple stressors that can affect them both directly and indirectly. By modifying either the composition of the community or the physiology of the microorganisms, press stressors may indirectly impact the ability of the biofilms to cope with disturbances. Extracellular polymeric substances (EPS) produced by the biofilm are known to play an important role in its resilience to various stresses. The aim of this study was to decipher to what extent slight modifications of environmental conditions could alter the resilience of phototrophic biofilm EPS to a realistic sequential disturbance (4-day copper exposure followed by a 14-day dry period). By using very simplified biofilms with a single algal strain, we focused solely on physiological effects. The biofilms, composed by the non-axenic strains of a green alga ( Uronema confervicolum ) or a diatom ( Nitzschia palea ) were grown in artificial channels in six different conditions of light intensity, temperature and phosphorous concentration. EPS quantity (total organic carbon) and quality (ratio protein/polysaccharide, PN/PS) were measured before and at the end of the disturbance, and after a 14-day rewetting period. The diatom biofilm accumulated more biomass at the highest temperature, with lower EPS content and lower PN/PS ratio while green alga biofilm accumulated more biomass at the highest light condition with lower EPS content and lower PN/PS ratio. Temperature, light intensity, and P concentration significantly modified the resistance and/or recovery of EPS quality and quantity, differently for the two biofilms. An increase in light intensity, which had effect neither on the diatom biofilm growth nor on EPS production before disturbance, increased the resistance of EPS quantity and the resilience of EPS quality. These results emphasize the importance of considering the modulation of community resilience ability by environmental conditions, which remains scarce in the literature., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Loustau, Leflaive, Boscus, Amalric, Ferriol, Oleinikova, Pokrovsky, Girbal-Neuhauser and Rols.)

Aim: Study of the biocidal effect of a cold atmospheric-pressure plasma in ambient air on single-species bacterial biofilms with controlled cell density, characterized by different extracellular matrices., Methods and Results: Two bacterial strains were chosen to present different Gram properties and contrasted extracellular matrices: Pseudomonas aeruginosa ATCC 15442 (Gram-negative), and Leuconostoc citreum NRRL B-1299 (Gram-positive). P. aeruginosa biofilm exhibits a complex matrix, rich in proteins while L. citreum presents the specificity to produce glucan-type exopolysaccharides when grown in the presence of sucrose. Plasma was applied on both surface-spread cells and 24-h grown biofilms with controlled cell loads over 5, 10, or 20 min. Surface-spread bacteria showed a time dependent response, with a maximal bacterial reduction of 2.5 log after 20 min of treatment. On the other hand, in our experimental conditions, no bactericidal effect could be observed when treating biofilms of P. aeruginosa and glucan-rich L. citreum ., Conclusions: For biofilms presenting equivalent cell loads, the response to plasma treatment seemed to depend on the properties of the extracellular matrix characterized by infrared spectroscopy, scanning electron microscopy, or dry weight., Significance and Impact of Study: Both cell load standardization and biofilm characterization are paramount factors to consider the biocide effect of plasma treatments. The extracellular matrix could affect the plasma efficacy by physical and/or chemical protective effects.

Background: Sugarcane distillery waste water (SDW) or vinasse is the residual liquid waste generated during sugarcane molasses fermentation and alcohol distillation. Worldwide, this effluent is responsible for serious environmental issues. In Reunion Island, between 100 and 200 thousand tons of SDW are produced each year by the three local distilleries. In this study, the potential of Aspergillus niger to reduce the pollution load of SDW and to produce interesting metabolites has been investigated., Results: The fungal biomass yield was 35 g L -1 corresponding to a yield of 0.47 g of biomass/g of vinasse without nutrient complementation. Analysis of sugar consumption indicated that mono-carbohydrates were initially released from residual polysaccharides and then gradually consumed until complete exhaustion. The high biomass yield likely arises from polysaccharides that are hydrolysed prior to be assimilated as monosaccharides and from organic acids and other complex compounds that provided additional C-sources for growth. Comparison of the size exclusion chromatography profiles of raw and pre-treated vinasse confirmed the conversion of humic- and/or phenolic-like molecules into protein-like metabolites. As a consequence, chemical oxygen demand of vinasse decreased by 53%. Interestingly, analysis of intracellular lipids of the biomass revealed high content in oleic acid and physical properties relevant for biodiesel application., Conclusions: The soft-rot fungus A. niger demonstrated a great ability to grow on vinasse and to degrade this complex and hostile medium. The high biomass production is accompanied by a utilization of carbon sources like residual carbohydrates, organic acids and more complex molecules such as melanoidins. We also showed that intracellular lipids from fungal biomass can efficiently be exploited into biodiesel.

This study aims to clarify the biochemical nature and interactions of Extracellular Polymeric Substances (EPS) involved in the structure and cohesive properties of aerobic granules. Granules were incubated with selective hydrolytic enzymes or with chemicals and the resistance of digested granules to shear stress was evaluated. After α-amylase digestion, the hydrodynamic stress released macro-particles (>315 μm) while soluble molecules (<1.5 μm) and micro-particles (1.5-315 μm) where mainly recovered after savinase and EDTA treatments. These data show that α (1-4) glucans and proteins are key polymers for granule cohesion and that divalent cationic bridging is a major aggregative mechanism. On the basis of these experiments and microscopy observations, a model is proposed for the spatial organization of EPS in the granular structure, in which α glucans are arranged in a capsular layer surrounding bacterial clusters while anionic proteins constitute the intercellular cement that may reinforce cohesion inside the bacterial clusters., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the alpha and beta subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250).

Background: The so-called antikeratin antibodies and the antiperinuclear factor are the most specific serological markers of rheumatoid arthritis (RA). They were recently shown to be largely the same autoantibodies and to recognize human epidermal filaggrins and profilaggrin-related proteins of buccal epithelial cells (collectively referred to as (pro)filaggrin)., Materials and Methods: To further characterize the target antigens, we investigated their expression by normal human epidermal keratinocytes cultured in differentiating conditions, using immunofluorescence and immunoblotting with RA sera and three different monoclonal antibodies to (pro)filaggrin., Results: On the cornified, stratified epithelial sheets obtained in vitro, RA sera with anti(pro)filaggrin autoantibodies (AFA) produced granular staining of the stratum granulosum and diffuse staining of the stratum corneum. The antigens recognized by RA sera strictly colocalized with (pro)filaggrin in keratohyalin granules. Following sequential extraction of the proteins from the epithelial sheets, the RA sera and the three monoclonal antibodies to (pro)filaggrin, recognized a series of low-salt-soluble molecules, including a neutral/acidic isoform of filaggrin and several proteins with sizes and pI intermediates between this isoform and profilaggrin. They also recognized urea-soluble high-molecular-weight profilaggrin-related molecules., Conclusions: These results show that in vitro epidermal keratinocytes express various molecular forms of (pro) filaggrin that bear epitopes targeted by AFA of RA sera, and that some of these are absent from epidermis. Moreover, these epitopes, which are present on the keratohyalin granules of buccal epithelial cells but not on those of epidermal cells, are present on the granules of the cultured keratinocytes. This work completes the molecular characterization of the proteins targeted by AFA.

Cornified cell envelope (CE) is generated during the late stages of epidermal differentiation and is made up of proteins covalently linked together by transglutaminases. To determine whether filaggrin is a component of this structure in humans, we analysed highly purified CE from plantar stratum corneum. An immunoelectron microscopy analysis showed specific binding of four different anti-(pro)filaggrin monoclonal antibodies to the surface of the CE, proved previously to be free of non-covalently linked proteins. Moreover, the anti-filaggrin activity of one of the antibodies was absorbed by preincubation with the plantar CE, as determined by ELISA. Convincingly, fragments of CE produced by proteolytic digestion of the structures were stained by this antibody on immunoblots. These data provide direct evidence that filaggrin is a component of CE purified from human plantar stratum corneum. Cross-linking between CE and the filaggrin-containing fibrous matrix may enhance the structural cohesion of the corneocytes and thus the resistance of the stratum corneum.

To improve understanding of human profilaggrin processing to filaggrin, we produced seven monoclonal antibodies against epidermal filaggrin (AHF1-7). They were characterized on human epidermis by indirect immunofluorescence, immunogold labeling, and immunoblotting and found to be directed against seven different epitopes of (pro)filaggrin. AHF1-5 labeled the keratohyalin granules and the fibrous matrix of the lower corneocytes, and recognized filaggrin and profilaggrin. AHF6 also labeled the keratohyalin granules and the corneocyte matrix, but only recognized filaggrin. In addition to this reactivity within the upper epidermis, AHF4-6 stained the cytoplasm of the basal cells, and cross-reactivity of AHF5 and AHF6 with cytokeratin K14 was revealed on immunoblots. It is interesting that AHF7 recognized filaggrin, but not profilaggrin, and labeled only the corneocyte matrix and not the keratohyalin granules. This indicates that filaggrin and cytokeratins share several antigenic determinants and that filaggrin bears at least one epitope absent from its precursor. The original series of monoclonal antibodies described here appears to be a powerful tool for studying human profilaggrin processing in normal conditions and in the keratinization disorders in which processing is altered.