Tânia F.C.V. Silva

Multistage treatment system for raw leachate from sanitary landfill combining biological nitrification–denitrification/solar photo-Fenton/biological processes, at a scale close to industrial – Biodegradability enhancement and evolution profile of trace pollutants

A multistage treatment system, at a scale close to the industrial, was designed for the treatment of a mature raw landfill leachate, including: a) an activated sludge biological oxidation (ASBO), under aerobic and anoxic conditions; b) a solar photo-Fenton process, enhancing the bio-treated leachate biodegradability, with and without sludge removal after acidification; and c) a final polishing step, with further ASBO. The raw leachate was characterized by a high concentration of humic substances (HS) (1211 mg CHS/L), representing 39% of the dissolved organic carbon (DOC) content, and a high nitrogen content, mainly in the form of ammonium nitrogen (>3.8 g NH4(+)-N/L). In the first biological oxidation step, a 95% removal of total nitrogen and a 39% mineralization in terms of DOC were achieved, remaining only the recalcitrant fraction, mainly attributed to HS (57% of DOC). Under aerobic conditions, the highest nitrification rate obtained was 8.2 mg NH4(+)-N/h/g of volatile suspended solids (VSS), and under anoxic conditions, the maximum denitrification rate obtained was 5.8 mg (NO2(-)-N + NO3(-)-N)/h/g VSS, with a C/N consumption ratio of 2.4 mg CH3OH/mg (NO2(-)-N + NO3(-)-N). The precipitation of humic acids (37% of HS) after acidification of the bio-treated leachate corresponds to a 96% DOC abatement. The amount of UV energy and H2O2 consumption during the photo-Fenton reaction was 30% higher in the experiment without sludge removal and, consequently, the reaction velocity was 30% lower. The phototreatment process led to the depletion of HS >80%, of low-molecular-weight carboxylate anions >70% and other organic micropollutants, thus resulting in a total biodegradability increase of >70%. The second biological oxidation allowed to obtain a final treated leachate in compliance with legal discharge limits regarding water bodies (with the exception of sulfate ions), considering the experiment without sludge. Finally, the high efficiency of the overall treatment process was further reinforced by the total removal percentages attained for the identified organic trace contaminants (>90%).

Biodegradability enhancement of a leachate after biological lagooning using a solar driven photo-Fenton reaction, and further combination with an activated sludge biological process, at pre-industrial scale

This work proposes an integrated leachate treatment strategy, combining a solar photo-Fenton reaction, to enhance the biodegradability of the leachate from an aerated lagoon, with an activated sludge process, under aerobic and anoxic conditions, to achieve COD target values and nitrogen content according to the legislation. The efficiency and performance of the photo-Fenton reaction, concerning a sludge removal step after acidification, defining the optimum phototreatment time to reach a biodegradable wastewater that can be further oxidized in a biological reactor and, activation sludge biological process, defining the nitrification and denitrification reaction rates, alkalinity balance and methanol dose necessary as external carbon source, was evaluated in the integrated system at a scale close to industrial. The pre-industrial plant presents a photocatalytic system with 39.52 m(2) of compound parabolic collectors (CPCs) and 2 m(3) recirculation tank and, an activated sludge biological reactor with 3 m(3) capacity. Leachate biodegradability enhancement by means of a solar driven photo-Fenton process was evaluated using direct biodegradability tests, as Zahn-Wellens method, and indirect measure according to average oxidation state (AOS), low molecular carboxylic acids content (fast biodegradable character) and humic substances (recalcitrant character) concentration. Due to high variability of leachate composition, UV absorbance on-line measurement was established as a useful parameter for photo-Fenton reaction control.

An innovative multistage treatment system for sanitary landfill leachate depuration: Studies at pilot-scale

In this work, an innovative methodology for the treatment of landfill leachates, after aerobic lagooning, is proposed and adjusted at pilot-scale. This methodology involves an aerobic activated sludge biological pre-oxidation (ASBO), a coagulation/sedimentation step (240mgFe3+/L, at pH4.2) and a photo-oxidation through a photo-Fenton (PF) reaction (60mg Fe2+, at pH2.8) combining solar and artificial light. The ASBO process applied to a leachate after aerobic lagooning, with high organic and nitrogen content (1.1-1.5gC/L; 0.8-3.0gN/L) and low biodegradability (BOD5/COD =0.07-0.13), is capable to oxidise 62-99% of the ammonium nitrogen, consuming only the affluent alkalinity (70-100%). The coagulation/sedimentation stage led to the humic acids precipitation, promoting a marked change in leachate colour, from dark-brown to yellowish-brown (related to fulvic acids), accompanied by a reduction of 60%, 58% and 88% on DOC, COD and TSS, respectively. The PF system promoted the degradation of the recalcitrant organic molecules into more easily biodegradable ones. According to Zahn-Wellens biodegradability test, a leachate with 419mg DOC/L after coagulation, would have to be photo-oxidized until DOC <256mg/L, consuming 117mM of H2O2 and 10.4kJ/L of accumulated UV energy, to achieve an effluent that can be biologically treated in compliance with the COD discharge limit (150mg O2/L) into water bodies. The biological process downstream from the photocatalytic system would promote a mineralization >60%. The PF step cost to treat 100m3/day of leachate was 6.41€/m3, combining 1339m2 of CPCs with 31 lamps.

In Vitro Anti-Inflammatory and Cytotoxic Effects of Aqueous Extracts from the Edible Sea Anemones Anemonia sulcata and Actinia equina

Marine invertebrates have been attracting the attention of researchers for their application in nutrition, agriculture, and the pharmaceutical industry, among others. Concerning sea anemones (Cnidaria), little is known regarding their metabolic profiles and potential value as a source of pharmacologically-active agents. In this work, the chemical profiles of two species of sea anemones Actinia equina and Anemonia sulcata, were studied by high-performance liquid chromatography with diode-array detection (HPLC-DAD) and its impact upon immune and gastric cells was evaluated. In both species, the methylpyridinium alkaloid homarine was the major compound in aqueous extracts. The extracts were effective in reducing lipopolysaccharide (LPS)-induced levels of nitric oxide (NO) and intracellular reactive oxygen species (ROS) in a macrophage model of inflammation. Both the extracts and the alkaloid homarine were effective in inhibiting phospholipase A2 (PLA2), a pivotal enzyme in the initial steps of the inflammatory cascade. In order to mimic the oral consumption of these extracts; their effect upon human gastric cells was evaluated. While no caspase-9 activation was detected, the fact that the endoplasmic reticulum-resident caspase-4, and also caspase-3, were activated points to a non-classical mechanism of apoptosis in human gastric cells. This work provides new insights on the toxicity and biological potential of sea anemones increasingly present in human nutrition.

Nitrogen Removal from Landfill Leachate by Microalgae.

Landfill leachates result from the degradation of solid residues in sanitary landfills, thus presenting a high variability in terms of composition. Normally, these effluents are characterized by high ammoniacal-nitrogen (N–NH4+) concentrations, high chemical oxygen demands and low phosphorus concentrations. The development of effective treatment strategies becomes difficult, posing a serious problem to the environment. Phycoremediation appears to be a suitable alternative for the treatment of landfill leachates. In this study, the potential of Chlorella vulgaris for biomass production and nutrients (mainly nitrogen and phosphorus) removal from different compositions of a landfill leachate was evaluated. Since microalgae also require phosphorus for their growth, different loads of this nutrient were evaluated, giving the following N:P ratios: 12:1, 23:1 and 35:1. The results have shown that C. vulgaris was able to grow in the different leachate compositions assessed. However, microalgal growth was higher in the cultures presenting the lowest N–NH4+ concentration. In terms of nutrients uptake, an effective removal of N–NH4+ and phosphorus was observed in all the experiments, especially in those supplied with phosphorus. Nevertheless, N–NO3− removal was considered almost negligible. These promising results constitute important findings in the development of a bioremediation technology for the treatment of landfill leachates.

Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium

The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis, M. leprae, M. avium, etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we show that peptides derived from bovine lactoferricin (LFcin) improve the antimicrobial activity of ethambutol against Mycobacterium avium growing inside macrophages. Moreover, the d-enantiomer of a short version of lactoferricin containing amino acids 17 to 30 (d-LFcin17–30) causes intramacrophagic death of M. avium by increasing the formation of lysosomes and autophagosomes. This work opens the way to the use of lactoferricin-derived peptides to treat infections caused by mycobacteria and highlights important modulatory effects of d-FLcin17–30 on macrophages, which may be useful under other conditions in which macrophage activation is needed. ABSTRACT Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17–30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17–30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17–30 did not localize to M. avium-harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17–30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis, M. leprae, M. avium, etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we show that peptides derived from bovine lactoferricin (LFcin) improve the antimicrobial activity of ethambutol against Mycobacterium avium growing inside macrophages. Moreover, the d-enantiomer of a short version of lactoferricin containing amino acids 17 to 30 (d-LFcin17–30) causes intramacrophagic death of M. avium by increasing the formation of lysosomes and autophagosomes. This work opens the way to the use of lactoferricin-derived peptides to treat infections caused by mycobacteria and highlights important modulatory effects of d-FLcin17–30 on macrophages, which may be useful under other conditions in which macrophage activation is needed.

Unravelling a Mechanism of Action for a Cecropin A-Melittin Hybrid Antimicrobial Peptide: The Induced Formation of Multilamellar Lipid Stacks

An understanding of the mechanism of action of antimicrobial peptides is fundamental to the development of new and more active antibiotics. In the present work, we use a wide range of techniques (SANS, SAXD, DSC, ITC, CD, and confocal and electron microscopy) in order to fully characterize the interaction of a cecropin A-melittin hybrid antimicrobial peptide, CA(1-7)M(2-9), of known antimicrobial activity, with a bacterial model membrane of POPE/POPG in an effort to unravel its mechanism of action. We found that CA(1-7)M(2-9) disrupts the vesicles, inducing membrane condensation and forming an onionlike structure of multilamellar stacks, held together by the intercalated peptides. SANS and SAXD revealed changes induced by the peptide in the lipid bilayer thickness and the bilayer stiffening in a tightly packed liquid-crystalline lamellar phase. The analysis of the observed abrupt changes in the repeat distance upon the phase transition to the gel state suggests the formation of an Lγ phase. To the extent of our knowledge, this is the first time that the Lγ phase is identified as part of the mechanism of action of antimicrobial peptides. The energetics of interaction depends on temperature, and ITC results indicate that CA(1-7)M(2-9) interacts with the outer leaflet. This further supports the idea of a surface interaction that leads to membrane condensation and not to pore formation. As a result, we propose that this peptide exerts its antimicrobial action against bacteria through extensive membrane disruption that leads to cell death.

Leaves and stem bark from Allophylus africanus P. Beauv.: An approach to anti-inflammatory properties and characterization of their flavonoid profile

The small tree Allophylus africanus, widespread in the African continent, has long been considered valuable, as noted by the number of reports on their multiple medicinal uses. With this work, we aimed to extend the current, and so far restricted, knowledge on the chemical composition of the plant, particularly as source of flavonoids, as well as to assess its potential anti-inflammatory properties. The chemical characterization of the aqueous extract obtained from the leaves allowed the identification and quantitation of 30 flavones, predominantly apigenin derivatives, but also four luteolin derivatives, while the stem bark extract was solely characterized by apigenin di-C-glycosides and mono-C-glycosides-O-glycosylated. Strong inhibitory effects towards 5-lipoxygenase were observed with the aqueous extracts obtained from the leaves and stem bark, with IC50 values of 41.28 and 107.77 μg mL-1, respectively. Both extracts were also found to reduce NO levels in LPS-challenged RAW 264.7 macrophages, without noticeable cytotoxicity. The flavonoid profile of the plant is disclosed for the first time, allowing the identification of several molecules that may contribute to mitigate the inflammatory response. Jointly, with the current study the anti-inflammatory use of the leaves and stem bark is partially validated.

Toxicity of phenolipids: Protocatechuic acid alkyl esters trigger disruption of mitochondrial membrane potential and caspase activation in macrophages

Phenolipids are a class of phenolic compounds with a lipidic moiety that have been receiving increasing attention due to their promising biological activities; however data regarding their toxicity and mechanism of action are scarce. A series of 11 phenolipids consisting of alkyl esters derivatives of the natural molecule protocatechuic acid was synthesized and evaluated against a panel of cancer and non-cancer cell lines. The macrophage cell line RAW 264.7, widely used as a tool for screening anti-inflammatory drugs, was more susceptible to the toxicity of these molecules than human cancer cells, reason for which mechanist studies were conducted. The parent molecule was not toxic up to 100μM, however structural modifications by inclusion of carbon side chains resulted in increased toxicity, compounds bearing 8-14 carbons being the most toxic and displaying IC50 in the nanomolar range. Mechanistic studies showed that phenolipids elicit chromatin condensation, loss of cell viability and disruption of mitochondrial membrane potential (ΔΨm), increased reactive oxygen species (ROS) and activation of caspase-9/3, thus pointing to the involvement of mitochondria in the programmed cell death process taking place. This is the first study addressing the toxicity and mechanism of action of protocatechuic acid derivatives, which is relevant in light of the recent interest in these molecules.