Effect of Chlorination on Microbiological Quality of Effluent of a Full-Scale Wastewater Treatment Plant.

The evaluation of effluent wastewater quality mainly relies on the assessment of conventional bacterial indicators, such as fecal coliforms and enterococci; however, little is known about opportunistic pathogens, which can resist chlorination and may be transmitted in aquatic environments. In contrast to conventional microbiological methods, high-throughput molecular techniques can provide an accurate evaluation of effluent quality, although a limited number of studies have been performed in this direction. In this work, high-throughput amplicon sequencing was employed to assess the effectiveness of chlorination as a disinfection method for secondary effluents. Common inhabitants of the intestinal tract, such as Bacteroides, Arcobacter and Clostridium, and activated sludge denitrifiers capable of forming biofilms, such as Acidovorax, Pseudomonas and Thauera, were identified in the chlorinated effluent. Chloroflexi with dechlorination capability and the bacteria involved in enhanced biological phosphorus removal, i.e., Candidatus Accumulibacter and Candidatus Competibacter, were also found to resist chlorination. No detection of Escherichia indicates the lack of fecal coliform contamination. Mycobacterium spp. were absent in the chlorinated effluent, whereas toxin-producing cyanobacteria of the genera Anabaena and Microcystis were identified in low abundances. Chlorination significantly affected the filamentous bacteria Nocardioides and Gordonia, whereas Zoogloea proliferated in the disinfected effluent. Moreover, perchlorate/chlorate- and organochlorine-reducing bacteria resisted chlorination.

Biotreatment efficiency, hydrolytic potential and bacterial community dynamics in an immobilized cell bioreactor treating caper processing wastewater under highly saline conditions.

Although caper processing wastewaters (CPW) are characterized by high organic content and salt concentration, no attempt has been made to treat these effluents. In this study, an immobilized cell bioreactor efficiently treated CPW even at hypersaline conditions (100 g/L salinity). Nitrogen was mainly assimilated during biotreatment, as nitrification was inhibited at elevated salinities. The hydrolytic potential was assessed by determining glucanase, xylanase, glucosidase, lipase and protease activities, which were negatively affected above 20 g/L salinity as the consequence of the inhibition of non-halotolerant microbiota. Succession of non-halotolerant taxa by the slightly halotolerant bacteria Defluviimonas, Amaricoccus, Arenibacter, Formosa and Muricauda, and then by the moderately/extremely halotolerant genera Halomonas, Roseovarius and Idiomarina occurred over salinity increase. Diversity indices were reduced during transition from moderately saline to hypersaline conditions. A distinct network was formed at hypersaline conditions, consisting of the halotolerant genera Halomonas, Idiomarina, Saliterribacillus and Gracilibacillus.

Biochemical and nutritional characterization of the medfly gut symbiont Enterobacter sp. AA26 for its use as probiotics in sterile insect technique applications.

BACKGROUND: Enterobacter sp. AA26 was recently isolated from the midgut of Ceratitis capitata (Wiedemann) and it was shown to have positive effects in rearing efficiency when used as larval probiotics. In this study, biomass production was carried out in bench-scale bioreactors to elucidate the biokinetic properties of Enterobacter sp. AA26 and its nutritional value. RESULTS: Strain AA26 is a psychrotolerant, halotolerant, facultatively anaerobic bacterium with broad pH range for growth (pH 4 to 10.2), which possessed the typical biochemical profile of Enterobacter spp. The specific oxygen uptake rate (SOUR) was calculated as 63.2 ± 1.26 and 121 ± 1.73 mg O2 g- 1 VSS h- 1, with the yield coefficients in acetate and glucose being equal to 0.62 ± 0.03 and 0.67 ± 0.003 g biomass produced/g substrate consumed, respectively. The maximum specific growth rate (µmax) of strain AA26 grown in fill-and-draw bioreactors at 20 °C and 35 °C was 0.035 and 0.069 h- 1, respectively. Strain AA26 grew effectively in agro-industrial wastewaters, i.e. cheese whey wastewater (CWW), as alternative substrate for replacing yeast-based media. Biomass of strain AA26 could provide all the essential amino acids and vitamins for the artificial rearing of C. capitata. Greater intracellular α- and ß-glucosidase activities were observed during growth of strain AA26 in CWW than in yeast-based substrate, although the opposite pattern was observed for the respective extracellular activities (p < 0.01). Low protease activity was exhibited in cells grown in yeast-based medium, while no lipase activities were detected. CONCLUSIONS: The ability of strain AA26 to grow in agro-industrial wastes and to provide all the essential nutrients can minimize the cost of commercial media used for mass rearing and large scale sterile insect technique applications.

Laser micro-structured Si scaffold-implantable vaccines against Salmonella Typhimurium.

Salmonella Typhi is responsible for typhoid fever in humans. Despite the efforts, the development of long-lasting vaccines has failed and the available vaccines display only moderate activity, being considered as "international traveler's" vaccines. Taking advantage of the previously described implantable vaccine technology consisting on 3D laser-microstructured Si scaffolds loaded with antigen-seeded macrophages, the present study aimed to apply an antigenic stimulus of whole extracts of S. Typhimurium, which is the mouse analogue of the human Salmonella Typhi, and examine its ability to mount specific antibody response. After defining the experimental conditions for specific anti-S. Typhimurium IgG production in vitro, antigen-seeded macrophages loaded onto the 3D Si-scaffolds were implanted to mice, while parallel experiments used conventional Freund-complete-adjuvant vaccination protocols. The results showed that only the implantable vaccine protocol could mount a specific antibody response 14 days after implantation. The cytokine profile showed increase of IL-10 and IFN-γ in the case of implantable and conventional vaccination respectively, 7 days after implantation. Morphological studies on the excised scaffolds 14 days after implantation, showed the development of a well-structured adherent monolayer, establishing multiple contacts with lymphocytes in favor to immune response development. Based on the hypothesis that both stimulatory and suppressive components in the vaccination preparation, could affect the overall activity, peptidoglycan was applied as an antigen to the vaccination protocols. Surprisingly, peptidoglycan was shown to induce a mitogenic rather than specific immunogenic response. In this case, histological analysis of the excised scaffolds showed a restricted layer of adherent cells with cytoplasmic extensions, but hard to distinguish cell contacts with lymphocytes. Finally, the presented results showed a differential behavior of antigen presenting cells in accordance to the antigenic stimulus and consequently the activation state of the cells. Tailoring the micro/sub-micron 3D structures and chemistry of Si scaffolds, could control cell behavior according to the user's needs.

Detection of the T cell activation state using nonlinear optical microscopy.

The ability to monitor the activation state of T-cells during immunotherapy is of great importance. Although specific activation markers do exist, their abundance and complicated regulation cannot definitely define the activation state of the cells. Previous studies have shown that Third Harmonic Generation (THG) imaging could distinguish between activated versus resting microglia and healthy versus cancerous cells, mainly based on their lipid-body profiles. In the present study, mitogen or antigen-stimulated T-cells were subjected to THG imaging microscopy. Qualitative and quantitative analysis showed statistically significant increase of THG mean area and intensity in activated versus resting T-cells. The connection of THG imaging to chemical information was achieved using Raman spectroscopy, which showed significant differences between the activation processes and controls, correlating of THG signal area with cholesterol and lipid compounds, but not with triglycerides. The obtained results suggested a potential employment of nonlinear microscopy in evaluating of T-cell activation, which is expected to be largely appreciated in the clinical practice.

Protection Against Lipopolysaccharide-Induced Immunosuppression by IgG and IgM.

Lipopolysaccharide (LPS) is commonly used in murine sepsis models, which are largely associated with immunosuppression and collapse of the immune system. After adapting the LPS treatment to the needs of locally bred BALB/c mice, the present study explored the potential role of IgG and IgM in reversing LPS endotoxemia. The established protocol consisted of five daily intraperitoneal injections of 0.2 µg/g LPS, which was tolerable by half of the manipulated animals. Such a protocol allowed longer survival, necessary in the prospect of therapeutic treatment application. This treatment significantly decreased CD4+, CD8+, CD3z+, and CD19+ cells, while increasing myeloid-derived suppressor cells (MDSCs; CD11b+Gr1+), CD25+ and Foxp3+ cells. These results were accompanied by increased arginase-1 activity in spleen cell lysates and production of IL-6, TNF-α, IL-18, and C-reactive protein (CRP) in the serum. The applied LPS protocol did not alter serum procalcitonin levels. MDSCs isolated from the spleen of LPS-treated animals (LPS-MDSCs) decreased proliferation of naive T cells in coculture experiments. The application of IgG and IgM to the naive T cell/LPS-MDSCs cocultures significantly decreased CD25+, Foxp3+, and CD3z+ cells, indicating an anti-suppressive effect of immunoglobulins. The in vivo application of IgG and IgM significantly decreased the percent of CD11b+Gr1+, CD25+, Foxp3+ cells, and arginase-1 activity in the spleen of LPS-treated animals, while decreasing IL-6, TNF-α, and CRP levels in the serum, allowing survival to all animals tested. In conclusion, these results reveal a novel mode of action of IgG/IgM in LPS endotoxemia, strengthening thus the use of immunoglobulin treatment is septic patients.

Dominance of candidate Saccharibacteria in a membrane bioreactor treating medium age landfill leachate: Effects of organic load on microbial communities, hydrolytic potential and extracellular polymeric substances.

A membrane bioreactor (MBR), accomplishing high nitrogen removal efficiencies, was evaluated under various landfill leachate concentrations (50, 75 and 100% v/v). Proteinous and carbohydrate extracellular polymeric substances (EPS) and soluble microbial product (SMP) were strongly correlated (p<0.01) with organic load, salinity and NH4+-N. Exceptionally high ß-glucosidase activities (6700-10,100Ug-1) were determined during MBR operation with 50% v/v leachate, as a result of the low organic carbon availability that extendedly induced ß-glucosidases to breakdown the least biodegradable organic fraction. Illumina sequencing revealed that candidate Saccharibacteria were dominant, independently of the leachate concentration applied, whereas other microbiota (21.2% of total reads) disappeared when undiluted leachate was used. Fungal taxa shifted from a Saccharomyces- to a newly-described Cryptomycota-based community with increasing leachate concentration. Indeed, this is the first report on the dominance of candidate Saccharibacteria and on the examination of their metabolic behavior in a bioreactor treating real wastewater.

Implantable vaccine development using in vitro antigen-pulsed macrophages absorbed on laser micro-structured Si scaffolds.

To overcome the limiting antigenic repertoire of protein sub-units and the side effects of adjuvants applied in second generation vaccines, the present work combined in vitro and in vivo manipulations to develop biomaterials allowing natural antigen-loading and presentation in vitro and further activation of the immune response in vivo. 3-dimensional laser micro-textured implantable Si-scaffolds supported mouse macrophage adherence, allowed natural seeding with human serum albumin (antigen) and specific antibody and inflammatory cytokine production in vitro. Implantation of Si-scaffolds loaded with antigen-activated macrophages induced an inflammatory reaction along with antigen-specific antibody production in vivo, which could be detected even 30 days post implantation. Analysis of implant histology using scanning electron microscopy showed that Si-scaffolds could be stable for a 6-month period. Such technology leads to personalized implantable vaccines, opening novel areas of research and treatment.

DOα⁻ß⁺ expression in favor of HLA-DR engagement in exosomes.

The expression of DOß and not DOα, in addition to the high intracellular DR, low DM levels and absence of surface DR expression in K562 and HL-60 cells introduce alternative regulatory pathways in DR trafficking and consequently the antigen presentation process. The present study attempted to define the naturally occurring DOα negative state and explain the role of DOß in the intracellular DR accumulation in K562 and HL-60 cells. Despite the absence of DOα, the DOß chain was detected in the endosomal compartments. The lack of DOα was found to be partially responsible for the absence of DR from the cell membrane since stable K562-DOα transfectants allowed expression of membrane DR. This expression could be significantly increased upon DM induction by IFN-γ, indicating that DM was another limiting factor for the migration of DR to the cell surface of K562 and HL-60 cells. Furthermore, intracellular DR co-localized with the exosome specific marker CD9, while culture supernatants were shown to contain exosome-engaged and exosome free DR activity as evaluated by SDS-page followed by western blot, ELISA and transmission electron microscopy analysis. These findings indicated that in DOα⁻ß⁺ cells, DR molecules were programmed to secretion rather than surface expression. The presented results provide novel regulatory processes as to DR trafficking, avoiding expression to the cell surface.