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Although the management of citrus residues is of concern, their valorization is a challenging alternative. In this work, raw orange juice processing wastewater was spontaneously fermented to determine the succession in microbial communities and identify new indigenous microbial strains of high pectinolytic activity. Pectinolytic strains were isolated using citrus pectin as growth substrate. A shift in fungal population observed during spontaneous fermentation, where the abundances of Hanseniaspora, Cystofilobasidium and Meyerozyma were significantly increased, whereas Pichia fermentans followed by Saccharomyces spp. became the predominant taxa in the fermented wastewater. A significant reduction in the relative abundance of homofermentative aerococci and an increase in the relative abundance of heterofermentative leuconostocs and lactobacilli occurred during fermentation, while enterococci were predominant throughout the fermentation process. Regarding ecological indices, opposite changes took place in the bacterial compared to fungal community structure, but in a less pronounced degree. Examination of beta-diversity revealed the key role of Pichia species in driving fungal community structure during fermentation. P. fermentans, the predominant taxon in the fermented wastewater, was found to be the most effective pectinolytic microorganism. Pectin hydrolysis, carbohydrate fermentation to ethanol, lactic acid and acetate, and citric acid fermentation were the most important factors, influencing microbial community structure.

Effective and side-effect-free vaccines are still difficult tasks to achieve for a great majority of antigenic stimuli. Pathogen manipulation to abort infectivity and antigen delivery to ensure immune responsiveness are the major components vaccine technology tries to resolve. However, the development of an immune response is still a complicated matter, lies on hundreds of parameters and any effort towards activation can easily lead to adverse effects, making immunotherapy very difficult to control. The present review attempts to highlight the major parameters affecting immune responsiveness and show that vaccine technology, except from pathogen manipulation and the development of antigen delivery systems, requires attention to additional check-points. Analyzing the recently described personalized implantable vaccine technology, it becomes obvious that the nature of each antigenic stimulus dictates different responsiveness to the organism, which discourages the use of universal adjuvant and antigen-delivery systems. On the contrary, the ex vivo tuning of the immune response proposed by the implantable vaccine technology, allows controllable amendment of the response. The development of personalized technologies is expected to provide valuable tools for the management of human pathology.

Sanitary landfilling is still considered worldwide as one of the most common methods applied for the management of the municipal solid waste. As a consequence, vast amounts of landfill leachate are generated annually, which are characterized by variability in physicochemical composition, owing to the stabilization process that occurs over the years. However, sustainable management of landfill leachate is a challenging issue, due to diverse chemical composition and high concentration in heavy metals and xenobiotics. Despite the fact that several studies have been reported on the biotreatment of landfill leachate, only in recent years has the microbial composition in such systems have been examined. In the present review, the key role of the microbial ecology involved in depurification and detoxification of landfill leachate in activated sludge and anaerobic systems is interpreted and ecological considerations influencing landfill leachate treatment are stated. Apart from the assessment of landfill toxicity on certain model organisms, this work provides an extensive overview on microbial communities performing key biological processes during landfill leachate treatment, including nitrification-denitrification, anammox and anaerobic digestion. Moreover, microbial aspects affecting nutrient removal efficiency in such biosystems are discussed.

Η πραγματοποίηση της παρούσας διδακτορικής διατριβής βασίστηκε στη διεξοδική διερεύνηση της φυλογενετικής και μεταβολικής ποικιλομορφίας των μικροβιακών κοινοτήτων που συμμετέχουν στην αποδόμηση αποβλήτων μεταποίησης αγροτικών προϊόντων, τόσο σε ανεπεξέργαστα απόβλητα ή σε απόβλητα που έχουν υποστεί αυθόρμητη ζύμωση, όσο και κατά τη βιολογική επεξεργασία αυτών σε βιοαντιδραστήρες ακινητοποιημένης βιομάζας. Η διερεύνηση αυτή εστιάστηκε σε υγρά απόβλητα και υπολείμματα των κυριότερων διεργασιών μεταποίησης του αγροτοβιομηχανικού τομέα, ήτοι απόβλητα χυμοποίησης πορτοκαλιών, τη μεταποίηση πιπεριάς και απόβλητα που προκύπτουν από την παρασκευή τουρσιού (κάπαρης). Η διερεύνηση των μικροβιακών κοινοτήτων διεξήχθη μέσω σύγχρονων τεχνικών μαζικής αλληλούχισης, αλλά και μέσω απομόνωσης νέων στελεχών μικροοργανισμών βιοτεχνολογικού ενδιαφέροντος, για την αξιοποίηση αυτών τόσο στην ενζυμική τεχνολογία και στην βιομηχανία τροφίμων και ποτών, όσο και στην αποδόμηση αγροτοβιομηχανικών αποβλήτων. Η εκτίμηση της υδρολυτικής ενεργότητας τόσο της μικτής βιομάζας, όσο και των απομονωμένων στελεχών μικροοργανισμών, μέσω προσδιορισμού της ενεργότητας των κυριότερων ενζύμων που εμπλέκονται στην αποδόμηση αγροτοβιομηχανικών υγρών αποβλήτων και υπολειμμάτων, επέτρεψε την αξιολόγηση της διαδικασίας βιοδιάσπασης των σύνθετων βιομορίων που περιλαμβάνονται στα απόβλητα της κατηγορίας αυτής. Για πρώτη φορά, τέθηκαν προς επεξεργασία σε βιολογικό σύστημα υγρά απόβλητα από τη μεταποίηση πιπεριάς, επετεύχθη δε αποτελεσματική αποδόμηση πραγματικού υγρού αποβλήτου που παρήχθη από τη μεταποίηση κάπαρης υπό υπερύαλες συνθήκες, δηλαδή υπό αλατότητα που υπερέβαινε το 10% w/v, και εκτενή πειραματική περίοδο. Επιπλέον, για πρώτη φορά πραγματοποιήθηκε ευρείας κλίμακας προσδιορισμός της υδρολυτικής ενεργότητας, και δη της μεταβολικής δραστηριότητας, αυτόχθονων μικροβιακών κοινοτήτων που εμβιούν σε απόβλητα που προκύπτουν από τη χυμοποίηση πορτοκαλιών. Η διδακτορική αυτή διατριβή εστίασε τόσο στις κοινότητες βακτηρίων, όσο και στις κοινότητες μυκήτων, και στο υδρολυτικό δυναμικό αυτών, για να αξιολογήσει σφαιρικά τις βιολογικές διεργασίες που λαμβάνουν χώρα κατά την αποδόμηση των κυριότερων αγροτοβιομηχανικών υπολειμμάτων. Η διδακτορική αυτή διατριβή, εκτός του θεωρητικού μέρους και της αναλυτικής περιγραφής των μεθόδων και τεχνικών που χρησιμοποιήθηκαν, περιλαμβάνει πέντε κεφάλαια αποτελεσμάτων με τη συζήτηση αυτών, ενώ το τελευταίο κεφάλαιο εστιάζει στη συνδυαστική αξιολόγηση και ανάδειξη των ευρημάτων αυτής. Αν και τα υγρά απόβλητα, τα οποία προέρχονται από την επεξεργασία της κάπαρης χαρακτηρίζονται από ιδιαίτερα υψηλή συγκέντρωση άλατος και οργανικών ενώσεων, εντούτοις, δεν υπάρχει στη διεθνή βιβλιογραφία έρευνα σχετικά με την επεξεργασία τους. Για το σκοπό αυτό, χρησιμοποιήθηκε αντιδραστήρας ακινητοποιημένης βιομάζας, ο οποίος λειτούργησε υπό υπερύαλες συνθήκες (100 g/L NaCl). Το υδρολυτικό δυναμικό της ακινητοποιημένης βιομάζας αξιολογήθηκε με τον προσδιορισμό της ενεργότητας των ενζύμων γλυκανάση, ξυλανάση, γλυκοσιδάση, λιπάση και πρωτεάση, οι οποίες επηρεάστηκαν αρνητικά σε συγκέντρωσεις άλατος μεγαλύτερες των 20 g/L. Οι ενεργότητες των ενζύμων εξω-1,4-β-γλυκανάση και β-1,4-ξυλοσιδάση, ακόμη και υπό αλατότητα 10% w/v, ήταν υψηλότερες από 265 ± 6,4 και 61,7 ± 2,7 U/g πρωτεΐνης, αντίστοιχα, η δε υψηλή συσχέτιση των ενεργοτήτων εξω-ξυλανασών και έξω-γλυκανασών (r=0,821, p

Ο ρόλος του ανοσοποιητικού συστήματος είναι η αναγνώριση και η καταπολέμηση των αντιγόνων με χυμικές και κυτταρικές ανοσοαποκρίσεις, με στόχο την εξάλειψη των ξένων ερεθισμάτων και την ανάπτυξη ειδικής μνήμης. Η ανάπτυξη ανοσοαπόκρισης έναντι ενός αντιγόνου έχει αξιοποιηθεί στο πλαίσιο των εμβολίων, τα οποία αποτελούν αντικείμενο ανάπτυξης για την πρόληψη ή βελτίωση των αποτελεσμάτων μελλοντικών παθογόνων μολύνσεων. Προκειμένου να αποφευχθεί η μολυσματικότητα, τα εμβόλια δεύτερης γενιάς κατασκευάστηκαν από καθορισμένα αντιγονικά πεπτίδια ή ανασυνδιασμένες υπομονάδες πρωτεινών, τα οποία όμως χρειάζονται ταυτόχρονη χορήγηση ανοσοενισχυτικού για την επαγωγή της ανοσοαπόκρισης.Ωστόσο, τα ανοσοενισχυτικά είναι υπεύθυνα για πολλές παρενέργειες και επιπλέον, λόγο του εκτεταμένου πολυμορφισμού των αντιγόνων ιστοσυμβατότητας, η επιλογή συγκεκριμένων μη λοιμωδών αντιγονικών επιτόπων μπορεί να μην είναι εξίσου αποτελεσματική για όλα τα άτομα. Για να ξεπεραστούν τα προβλήματα αυτά πραγματοποιήθηκε ανάπτυξη ενός συστήματος που να επιτρέπει τη φυσική "φόρτωση" και παρουσίαση αντιγόνων in vitro και την περαιτέρω ενεργοποίηση της ανοσολογικής απόκρισης in vivo.Μια τέτοια τεχνολογία οδηγεί σε εξατομικευμένα εμφυτεύσιμα εμβόλια, όπου μακροφάγα εμβολιάζονται με το αντιγόνο με φυσικό τρόπο και αφού ενεργοποιηθούν, προσκολλώνται σε εμφυτεύσιμες επιφάνειες και είναι σε θέση να ενισχύσουν την έναρξη ανοσολογικής απόκρισης. Μια τέτοια προσέγγιση εξαλείφει τις παρενέργειες που οφείλονται στη μη ειδική διέγερση των ανοσοενισχυτικών, αφήνοντας το φυσικό μηχανισμό επιλογής "φόρτωσης" αντιγόνου στα αντιγονοπαρουσιαστικά κύτταρα ώστε να επιλεγεί ο κατάλληλος αντιγονικός επίτοπος για κάθε άτομο. Οι εμφυτεύσιμες επιφάνειες αποτελούνται από ικριώματα πυριτίου με τρισδιάστατη μικρο- και νανο- υφή και κατασκευάζονται με τη χρήση λέιζερ υπερταχεών παλμών. Η εφαρμογή της τεχνολογίας αυτής σε ανθρώπους θα είναι μεγάλης σημασίας για το άνοιγμα νέων τομέων στην έρευνα και τη θεραπεία.

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.

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 β-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 ). 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.

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

The orange juice processing sector produces worldwide massive amounts of waste, which is characterized by high lignin, cellulose and hemicellulose content, and which exceeds 40% of the fruit&rsquo, s dry weight (d.w.). In this work, the diversity and the biotechnological potential of xylan-degrading microbiota in orange juice processing waste were investigated through the implementation of an enrichment isolation strategy followed by enzyme assays for the determination of xylanolytic activities, and via next generation sequencing for microbial diversity identification. Intracellular rather than extracellular endo-1,4-&beta, xylanase activities were detected, indicating that peripheral cell-bound (surface) xylanases are involved in xylan hydrolysis by the examined microbial strains. Among the isolated microbial strains, bacterial isolates belonging to Pseudomonas psychrotolerans/P. oryzihabitans spectrum (99.9%/99.8% similarity, respectively) exhibited activities of 280 U/mg protein. In contrast, almost all microbial strains isolated exerted low extracellular 1,4-&beta, xylosidase activities (&lt, 5 U/mg protein), whereas no intracellular 1,4-&beta, xylosidase activities were detected for any of them. Illumina data showed the dominance of lactic and acetic acid bacteria and of the yeasts Hanseniaspora and Zygosaccharomyces. This is the first report on indigenous xylanolytic microbiota isolated from orange juice processing waste, possessing the biotechnological potential to serve as biocatalysts for citrus biomass valorization through the production of high-added value products and energy recovery.

Wastewater treatment plants (WWTPs) highly contribute to the transmission of antibiotic resistance genes (ARGs) in the environment. In this work, the diversity of ermF, ermB, sul1 and int1-enconding genes was examined in the influent, the mixed liquor and the effluent of a full-scale WWTP. Based on the clones analyzed, similar genotypes were recorded at all process stages. However, distinct genotypes of int1 were responsible for the expression of sul1 and ermF genes in Gammaproteobacteria and Bacteroidetes, respectively. Due to the detection of similar ARGs profiles throughout the biological process, it is concluded that additional treatment is needed for their retention.

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.

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.

Third Harmonic Generation (THG) imaging was applied to mouse resting and activated T-cells. Quantification of THG signal, which corresponded to lipid droplets, could distinguish activated Tcells, allowing follow-up of immune response development.

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.

The expression of DOβ 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 DOβ in the intracellular DR accumulation in K562 and HL-60 cells. Despite the absence of DOα, the DOβ 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⁻β⁺ 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.

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.

Biomaterial surface properties are determined by the synergistic effect of the morphology and chemistry. Specific combinations of chemical and topographical cues, similar to those experienced by cells during in vivo development, have been shown to control cellular adhesion, migration and function. The aim of the study was to investigate whether 3D micro and submicron laser textured transplantable Si scaffolds with tunable morphology and chemistry could support macrophage adherence, antigen presentation and specific antibody production. The 3D scaffolds could provide useful materials for in vitro and in vivo applications.