Unidade de Investigação – LSRE-LCM – Laboratório de Processos de Separação e Reação – Laboratório de Catálise e Materiais – Polo IPLeiria
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O polo do LSRE-LCM – Laboratório de Processos de Separação e Reação – Laboratório de Catálise e Materiais do Politécnico de Leiria foi criado em 2011 e atualmente integra o maior Laboratório Associado Português em Engenharia Química, ALiCE, com uma intervenção muito relevante nas áreas de Engenharia do Ambiente e da Bioengenharia.
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- Adding value to stalked barnacles from Berlengas Nature Reserve (Portugal) by the development of a new foodPublication . Pinheiro, Joaquina; Fernandes, Wilson; Sá, Hugo; Bernardino, Raul; Leandro, Sérgio Miguel; Ganhão, RuiIn this work, a new and natural food product, stalked barnacle (Pollicipes Pollicipes) pâté enriched with blackberry, was developed to valorise the rejected stalked barnacle. To evaluate the addition of blackberry fruits (Rubus ulmifolius Schott.) as a natural preservative on pâté quality, four pâté sample groups were considered: a negative control without a synthetic additive (CTR), a positive control with butylated hydroxytoluene (BHT), a group with blackberry extract (blackberry), and a group with a mixture of BHT and blackberry (blackberry + BHT). In addition, the effect of pasteurization (80 °C for 30 min) versus sterilization (121 °C for 30 min) on the pâté quality were evaluated. The bioactive evaluation expressed by the total phenolic content (TPC) and antiradical activity by the DPPH radical scavenging associated with oxidative stability determined by thiobarbituric acid reacting substances (TBARS) were performed. The impact of packaging opening followed by refrigerated storage (4 ± 1 °C) simulating the consumer behavior at home, was assessed. All the heat-treated stalked barnacle pâté samples were found microbially safe with an interesting content of total phenolic, the highest ones being the CTR and Blackberry + BHT, ranging from 58.79 to 55.38 mg GAE/100 g. After the package opening of the barnacle pâté sample of Blackberry + BHT, it revealed a superior inhibition of TBARS (0.46 mg MDA/100 g) after 7 days at refrigerated storage compared to the other samples. These results state the efficacy of blackberry in minimizing the lipid oxidation of stalked barnacle pâte. This study showed the potential for rejected stalked barnacle to be valorised and improve the sustainability of resources.
- Adsorption Behavior and Mechanism of Oxytetracycline on Rice Husk Ash: Kinetics, Equilibrium, and Thermodynamics of the ProcessPublication . Andrade, Christhel A.; Zambrano-Intriago, Luis Angel; Oliveira, Nelson S.; Vieira, Judite S.; Quiroz-Fernández, Luis Santiago; Rodríguez-Díaz, Joan ManuelThe main objective of the present study is to determine the kinetics, thermodynamics, and adsorption mechanism of the oxytetracycline (OTC) on rice husk ash (RHA). The adsorbent was characterized by scanning electronic microscopy, Fourier transform infrared spectroscopy, and nitrogen physisorption. Batch studies were carried out to evaluate the influence of the adsorbent dose, initial concentration, contact time, temperature, and initial pH. RHA was characterized as having heterogeneous, fibrous, and porous particles, consisting predominantly of silica. The removal of OTC depends on the pH of the medium, which is favored at acid pH values. The kinetic data followed the Bangham model, which indicated an OTC diffusion in the pores of RHA, although this was not the only process, as demonstrated through the use of the Weber-Morris model (IPD model). The Sips isotherm best represents the experimental results of the equilibrium study. It was found that the adsorption process was spontaneous and endothermic. The highest adsorption capacity was found at a pH in the range of 4–6, when the OTC is in its zwitterion form and the surface of the RHA is positively charged, thus permitting electrostatic interactions and the formation of hydrogen bonds between the adsorbent and adsorbate molecules. These findings demonstrate the potential of rice husk ash to remove oxytetracycline from water.
- Anaerobic Mesophilic Co-Digestion of Swine Slurry and Hidrolyzate in Batch Reactors: A Case StudyPublication . Domingues, Paulo S.; Pala, Helena; Oliveira, Nelson S.The management of residual flows from swine farms, such as slurry and hydrolyzate, is a serious environmental problem in Portugal, so the search for new solutions is important, especially if they can be implemented in production sites, avoiding the transport of waste. Anaerobic co-digestion can configure a sustainable method of management and valorization of these flows in swine farms, resulting in biogas to produce energy and a digestate with agronomic value. The swine hydrolyzate is the product from the elimination of swine carcasses on the farms, through a hydrolysis process. Its use in the anaerobic process wasn’t found in the literature, identifying the need to evaluate its potential. Thus, anaerobic co-digestion of swine slurry and hydrolyzate was carried out and the main purpose of this research was to find the best feeding ratio between the two substrates in batch test, focusing on biogas production. The study was developed in two phases, both under mesophilic conditions. In the first one, using 100 ml syringes, the effect of the relative proportion of substrates on the anaerobic co-digestion potential was investigated and the effect of pressure sterilization of the substrates on biogas production was verified. In the second phase, a larger scale study was carried out in a 4,500 ml digestor, ran under the conditions identified as the most favorable in the preliminary tests. The results obtained allowed us to conclude that the largest volume of accumulated biogas was obtained with the proportion of 90.9% swine slurry: 9.1% swine hydrolyzate (v/v); and that the sterilization of the substrates doesn’t constitute an effective thermal pre-treatment. The larger scale test revealed an inefficient anaerobic process due to the inhibitory effect caused by the accumulation of ammonia and volatile organic acids. However, the production yield was 606.8 LBiogas/kgVS and 431.6 LMethane/kgVS, indicating that process allowed a production of biogas and methane higher than the values cited in literature for anaerobic digestion of swine slurry. This result showed that the use of swine hydrolyzate as a co-substrate results in a better balance of nutrients, promoting a better development of microorganisms.
- Autosomal recessive hypotrichosis with loose anagen hairs associated with TKFC mutations*Publication . Onoufriadis, A.; Cabezas, A.; Ng, J. C. F.; Canales, J.; Costas, M. J.; Ribeiro, J. M.; Rodrigues, J. R.; McAleer, M. A.; Castelo‐Soccio, L.; Simpson, M. A.; Fraternali, F.; Irvine, A. D.; Cameselle, J. C.; McGrath, J. A.Background: Loose anagen hair is a rare form of impaired hair anchorage in which anagen hairs that lack inner and outer root sheaths can be gently and painlessly plucked from the scalp. This condition usually occurs in children and is often self-limiting. A genetic basis for the disorder has been suggested but not proven. A better understanding the aetiology of loose anagen hair may improve prevention and treatment strategies. Objectives: To identify a possible genetic basis of loose anagen hair using next-generation DNA sequencing and functional analysis of variants identified. Methods: In this case study, whole-exome sequencing analysis of a pedigree with one affected individual with features of loose anagen hair was performed. Results: The patient was found to be compound heterozygous for two single-nucleotide substitutions in TKFC resulting in the following missense mutations: c.574G> C (p.Gly192Arg) and c.682C> T (p.Arg228Trp). Structural analysis of human TKFC showed that both mutations are located near the active site cavity. Kinetic assays of recombinant proteins bearing either of these amino acid substitutions showed almost no dihydroxyacetone kinase or D-glyceraldehyde kinase activity, and FMN cyclase activity reduced to just 10% of wildtype catalytic activity. Conclusions: TKFC missense mutations may predispose to the development of loose anagen hairs. Identification of this new biochemical pathobiology expands the metabolic and genetic basis of hypotrichosis.
- A Combined Vermifiltration-Hydroponic System for Swine Wastewater TreatmentPublication . Ispolnov, Kirill; Aires, Luis M. I.; Lourenço, Nídia D.; Vieira, Judite S.Intensive swine farming causes strong local environmental impacts by generating effluents rich in solids, organic matter, nitrogen, phosphorus, and pathogenic bacteria. Insufficient treatment of hog farm effluents has been reported for common technologies, and vermifiltration is considered a promising treatment alternative that, however, requires additional processes to remove nitrate and phosphorus. This work aimed to study the use of vermifiltration with a downstream hydroponic culture to treat hog farm effluents. A treatment system comprising a vermifilter and a downstream deep-water culture hydroponic unit was built. The treated effluent was reused to dilute raw wastewater. Electrical conductivity, pH, and changes in BOD5, ammonia, nitrite, nitrate, phosphorus, and coliform bacteria were assessed. Plants were monitored throughout the experiment. Electrical conductivity increased due to vermifiltration; pH stayed within a neutral to mild alkaline range. Vermifiltration removed 83% of BOD5, 99% of ammonia and nitrite, and increased nitrate by 11%. Hydroponic treatment removed BOD5 (63%), ammonia (100%), nitrite (66%), nitrate (27%), and phosphorus (47% total and 44% dissolved) from vermifiltered water. Coliforms were reduced by vermifiltration but recovered in the hydroponic unit. Plants showed the ability to grow on vermi-filtered wastewater, although requiring nutrient supplementation. Vermifiltration combined with hydroponics is a promising treatment for swine wastewater, although optimization will be needed for a sustainable real-scale implementation.
- A Community-Based Participatory Framework to Co-Develop Patient Education Materials (PEMs) for Rare Diseases: A Model Transferable across DiseasesPublication . Falcão, Marta; Allocca, Mariateresa; Rodrigues, Ana Sofia; Granjo, Pedro; Francisco, Rita; Pascoal, Carlota; Rossi, Maria Grazia; Marques-da-Silva, Dorinda; Magrinho, Salvador C. M.; Jaeken, Jaak; Castro, Larisa Aragon; Freitas, Cláudia de; Videira, Paula A.; Andrés-Aguayo, Luísa de; Ferreira, Vanessa dos ReisAt least 50% of chronic disease patients don’t follow their care plans, leading to lower health outcomes and higher medical costs. Providing Patient Education Materials (PEMs) to individuals living with a disease can help to overcome these problems. PEMs are especially beneficial for people suffering from multisystemic and underrecognized diseases, such as rare diseases. Congenital disorders of glycosylation (CDG) are ultra-rare diseases, where a need was identified for PEMs in plain language that can clearly explain complex information. Community involvement in the design of PEMs is extremely important for diseases whose needs are underserved, such as rare diseases; however, attempts to involve lay and professional stakeholders are lacking. This paper presents a community-based participatory framework to co-create PEMs for CDG, that is transferable to other diseases. A literature review and questionnaire were performed, and only four articles describing the development of PEMS for rare diseases have been found, which demonstrates a lack of standardized approaches. The framework and PEMs were co-developed with CDG families and will be crucial in increasing health literacy and empowering families. We will close a gap in the creation of PEMs for CDG by delivering these resources in lay language in several languages.
- Comparison of cytochrome c with conventional biocatalysts in the degradation of environmental toxicantsPublication . Lagoa, Ricardo; Lopes, João M.; Rodrigues, J. Rui; Marques-da-Silva, DorindaPeroxidases and laccases are amongst the most attractive enzymes for the degradation of concerning pollutants like polycyclic aromatic hydrocarbons (PAHs) and organic dyes. In spite of the availability of high activity enzymes, their applicability is hindered by specificity and stability limitations in real environmental conditions, so more suitable biocatalysts are demanded. Cytochrome c (Cc) is best known as a protein electron carrier at mitochondria, but it also displays (pseudo-)peroxidase activity. In this work, we aimed to evaluate the potential of Cc as biocatalyst of PAHs’ and azo dyes’ degradation and compare it with two more conventional enzymes - plant peroxidases and fungi laccases. The studies were carried out with Cc from horse heart, horseradish peroxidase (HRP) and laccase from Trametes versicolor. The enzymes were tested with two major PAHs, anthracene and benzo[a]pyrene (BaP), and with methyl orange (MO) as a model azo dye. The enzyme-catalyzed oxidation of PAHs was determined by HPLC, and MO decolorization was followed in spectrophotometric kinetic assays. Several degradation studies were performed to assess the catalytic capacity at different pH and the effect of the redox mediator ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)). Peroxidase reactions of HRP and Cc were initiated with 100 μM H2O2 (in media containing 100 μM DTPA as metal chelator). In pH 5 acetate buffer, the specific ABTS-oxidizing activity of Cc was much lower than that of laccase and HRP. However, these two enzymes lost almost all activity at pH 7, while Cc exhibited catalytic activity even at pH 8 (phosphate buffer). Decolorization assays showed laccase alone to be a weak catalyst of MO degradation, although addition of ABTS to the reaction media greatly accelerated the transformation. On the contrary, both HRP and Cc directly catalyzed MO decolorization, including at neutral pH, and the redox mediator offered no advantage. As for the PAHs, HRP and laccase catalysis benefited from the presence of ABTS in the media, but again Cc oxidized anthracene and BaP directly. Indeed, 24h incubation of BaP (1 mg/L) with Cc (0.1 mg/mL) resulted in the transformation of 70±4% of the PAH and generation of major reaction products different of the BaP quinones produced by laccase- and HRP-ABTS systems. This work disclosed catalytic properties of Cc different from laccase and HRP that are relevant for the design of new enzymatic remediation processes.
- Comparison of Laccases and Hemeproteins Systems in Bioremediation of Organic PollutantsPublication . Lopes, João M.; Marques-da-Silva, Dorinda; Videira, Paula Q.; Lagoa, Ricardo L.Aim: Laccases and peroxidases have attracted great interest for industrial and environmental applications. These enzymes have a broad substrate range and a robust oxidizing ability. Moreover, using mediators or co-oxidants makes it possible to increase their catalytic activity and extend their substrate scope to more resistant chemical structures. Background: Fungal laccases and ligninolytic peroxidases, mainly lignin and manganese peroxidases, are the privileged oxidoreductases for bioremediation processes. Nonetheless, an increasing diversity of laccases and peroxidase-type enzymes has been proposed for environmental technologies. Objective: This article aims to provide an overview of these enzymes and compare their applicability in the degradation of organic pollutants. Methods: Fundamental properties of the proteins are covered and applications towards polycyclic aromatic hydrocarbons (PAHs) and pesticides are specially focused. Results: Laccases are multicopper oxidases initially studied for applications in the pulp and paper in-dustry but able to oxidize a variety of environmentally concerning compounds. Relying on O2, laccases do not require peroxides nor auxiliary agents, like Mn2+, although suitable redox mediators are needed to attack the more recalcitrant pollutants (e.g., PAHs). True and pseudo-peroxidases use a stronger oxi-dant (H2O2) and the redox chemistry at the heme site generates high potential species that allow the oxidation of dyes and some pesticides. Conclusion: Lately, research efforts have been directed to enzyme discovery, testing with micropollu-tants, and improving biocatalysts’ stability by immobilization and protein engineering. Further under-standing of the effects of natural media components and solvents on the enzymes might lead to compet-itive enzymatic treatments of highly toxic media.
- Complexation and DFT studies of lanthanide ions by (2-pyridylmethoxy)homooxacalixarene derivativesPublication . Marcos, Paula M.; Teixeira, Filipa A.; Segurado, Manuel A.P.; Ascenso, José R.; Bernardino, Raul; Cragg, Peter J.; Michel, Sylvia; Hubscher-Bruder, Véronique; Arnaud-Neu, FrançoiseThe binding of lanthanide cations by 2-pyridylmethoxy derivatives of p-tert-butyldihomooxacalix[4]arene (1b), in the cone conformation, and p-tert-butylhexahomotrioxacalix[3]arene (2b), in both cone and partial cone conformations, was studied. These properties were assessed by extraction studies of the metal picrates from water into dichloromethane and stability constant measurements in methanol and acetonitrile, using spectrophotometric and microcalorimetric techniques. Proton NMR titrations with La3+ and Yb3+ cations were done in order to get information on the binding sites. Computational methods (density functional theory (DFT) calculations) were also used to complement the NMR data. The p-tert-butylcalix[4]arene analogue (3b) was also studied, and the results of the four ligands were compared. Partial cone-2b is the best extractant for lanthanide ions, showing some preference for the heavy lanthanides. In complexation, all four ligands show the same trend and a high selectivity for Yb3+ (ML, log β ≥ 7). Besides the formation of ML complexes, ML2 species were also obtained. In most cases, these species were corroborated by the proton NMR studies. For partial cone-2b with Pr3+ the complexation process is enthalpically driven, whereas for 3b the formation of the ML2 species with this cation is due to a favourable entropy term. DFT studies indicate that ligand 3b forms the most stable complex with La3+, followed by partial cone-2b.
- Complexation and DFT studies of lower rim hexahomotrioxacalix[3]arene derivatives bearing pyridyl groups with transition and heavy metal cations. Cone versus partial cone conformationPublication . Marcos, Paula M.; Teixeira, Filipa A.; Segurado, Manuel A. P.; Ascenso, José R.; Bernardino, Raul; Peter J. Cragg; Sylvia Michel; Véronique Hubscher-Bruder; Françoise Arnaud-NeuThe binding of representative alkali, alkaline earth, transition and heavymetal cations by 2-pyridylmethoxy derivatives (1b, in cone and partial cone conformations) of p-tert-butylhexahomotrioxacalix[3]arene was studied. Binding was assessed by extraction studies of the metal picrates from water into dichloromethane and by stability constant measurements in acetonitrile and methanol, using spectrophotometric and potentiometric techniques. Microcalorimetric studies of some selected complexes in acetonitrile were performed, as well as proton NMR titrations. Computational methods (density functional theory calculations) were also employed to complement the NMR data. The results are compared with those obtained with the dihomooxacalix[4]arene 2b and the calix[4]arene 3b derivative analogues. Partial cone-1b is the best extractant for transition and heavy metal cations. Both conformers of 1b exhibit very high stability constants for soft and intermediate cations Pb2+, Cd2+, Hg2+, Zn2+ and Ni2+, with cone-1b the strongest binder (ML, log b ≥7) and partial cone-1b the most selective. Both derivatives show a slight preference for Na+. Besides the formation of ML complexes, ML2 and M2L species were also observed. The former complexes were, in general, formed with the transition and heavy metal cations, whereas the latter were obtained with Ag+ and Hg2+ and partial cone-1b. In most cases, these species were corroborated by the proton NMR and density functional theory studies.