Ana Rodrigues

Wound healing activity of the human antimicrobial peptide LL37

Antimicrobial peptides (AMPs) are part of the innate immune system and are generally defined as cationic, amphipathic peptides, with less than 50 amino acids, including multiple arginine and lysine residues. The human cathelicidin antimicrobial peptide LL37 can be found at different concentrations in many different cells, tissues and body fluids and has a broad spectrum of antimicrobial and immunomodulatory activities. The healing of wound is a complex process that involves different steps: hemostasis, inflammation, remodeling/granulation tissue formation and re-epithelialization. Inflammation and angiogenesis are two fundamental physiological conditions implicated in this process. We have recently developed a new method for the expression and purification of recombinant LL37. In this work, we show that the recombinant peptide P-LL37 with a N-terminus proline preserves its immunophysiological properties in vitro and in vivo. P-LL37 neutralized the activation of macrophages by lipopolysaccharide (LPS). Besides, the peptide induced proliferation, migration and tubule-like structures formation by endothelial cells. Wound healing experiments were performed in dexamethasone-treated mice to study the effect of LL37 on angiogenesis and wound regeneration. The topical application of synthetic and recombinant LL37 increased vascularization and re-epithelialization. Taken together, these results clearly demonstrate that LL37 may have a key role in wound regeneration through vascularization.

Feasibility of a pulsed Sequencing Batch Reactor with anaerobic aggregated biomass for the treatment of low strength wastewaters

This study concerns an assessment of a SBR operation that associates anaerobic aggregated biomass with a pulsed action during the reaction phase, a system named Pulsed Sequencing Batch Reactor (P-SBR). The system uses a diaphragm pump as a pulsator unit to increase the liquid-solid contact, in order to avoid dead zones and possible external mass transfer resistance. A preliminary study of the operation of the reactor was performed with a low strength synthetic wastewater with a COD near 1000 mg.1 −1 and a sub-optimal temperature of 22°C. A removal efficiency of 60-70% was attained after 5 and 6 hours of reaction time. The respective organic loads were 5 – 6 kg COD.m −3 . day −1 , thus supporting the feasibility of the P-SBR system for wastewater treatment in such conditions. The results also indicate that a ratio of 1.8%o between the swept volume delivered by the pump and the reactor volume was adequate to promote a flow turbulence in the sludge blanket and that a redox potential of near −400 mV was readily created by anaerobic bacteria after the reactor filling step.

Recycling of cellulases in lignocellulosic hydrolysates using alkaline elution

The recovery of cellulases from lignin, lignocellulosic hydrolysates and cellulose by alkaline washes at pH 9 and 10 was examined. The effect of the pH on the structural stability of purified Cel7A was analyzed by circular dichroism. Purified Cel7A showed conformational changes at pH 9 and 10 that were reversible at pH 4.8. Temperature influenced the enzymatic hydrolysis of wheat straw and may be critical for the efficiency of cellulase recycling from wheat straw hydrolysates. Operation at moderate temperatures (37°C) resulted in a rate of saccharification 19% higher than that obtained at 50° C, improving cellulase recycling by 49%. Over 60% of the enzyme activity on the synthetic substrate 4-methylumbelliferyl-β-d-cellobioside (MUC) may be recovered by using a simple alkaline wash. This is thus a promising strategy for enzyme recycling that is simple to implement at industrial scale, economical and effective.

Brewery and winery wastewater treatment : some focal points of design and operation

Environmental issues are a critical factor for the today industry competitiveness. Indeed, the society and the individual consumers could set a common framework for companies’ commitment and engagement regarding environment protection. Redesign the process, recover by-products or reuse effluents are some of the possible actions towards an eco-efficient strategy. Nevertheless, a point remains crucial in such mission: the ability to defend natural ecosystems from polluted wastewaters. For such purpose, a wastewater treatment plant that maximizes removal efficiency and minimizes investment and operation costs is a key factor. Brewery and winery are traditional industries with an important economic value in the agro-food sector. In 2003, the total beer production in the European Union (18 countries) was 344 x 10 m, being recorded around 1800 breweries with 110 thousand employees. If Norway, Switzerland and Turkey are also included, those numbers rise up to 358 x 10 m, 1839 units and 117 thousand, respectively. The excise revenue from beer industry in all these countries reaches over 8800 x 10 € (The Brewers of Europe, 2004). The worldwide wine production is 261 x 10 m (data from 2002), of which 69 % from Europe, 18 % from America, 5 % from Asia, 4 % from Africa and 4 % from Oceania. The worldwide wine consumption (2002) is 228 x 10 m, distributed by Europe (68 %), America (20 %), Asia (7 %), Africa (3 %) and Oceania (2 %) (OIV, 2002). This chapter intends to present some key points on design and operation in wastewater treatment of brewery and winery industries. Therefore, an introduction of the industrial processes is first presented and then wastewater characteristics and treatment processes are discussed. Finally, the experience of a collaborative effort between

Celluclast and Cellic® CTec2: Saccharification/fermentation of wheat straw, solid–liquid partition and potential of enzyme recycling by alkaline washing

The hydrolysis/fermentation of wheat straw and the adsorption/desorption/deactivation of cellulases were studied using Cellic(®) CTec2 (Cellic) and Celluclast mixed with Novozyme 188. The distribution of enzymes - cellobiohydrolase I (Cel7A), endoglucanase I (Cel7B) and β-glucosidase - of the two formulations between the residual substrate and supernatant during the course of enzymatic hydrolysis and fermentation was investigated. The potential of recyclability using alkaline wash was also studied. The efficiency of hydrolysis with an enzyme load of 10 FPU/g cellulose reached >98% using Cellic(®) CTec2, while for Celluclast a conversion of 52% and 81%, was observed without and with β-glucosidase supplementation, respectively. The decrease of Cellic(®) CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and β-glucosidase. The adsorption/desorption profiles during hydrolysis/fermentation revealed that a large fraction of active enzymes remained adsorbed to the solid residue throughout the process. Surprisingly, this was the case of Cel7A and β-glucosidase from Cellic, which remained adsorbed to the solid fraction along the entire process. Alkaline washing was used to recover the enzymes from the solid residue. This method allowed efficient recovery of Celluclast enzymes; however, this may be achieved only when minor amounts of cellulose remain present. Regarding the Cellic formulation, neither the presence of cellulose nor lignin restricted an efficient desorption of the enzymes at alkaline pH. This work shows that the recycling strategy must be customized for each particular formulation, since the enzymes found e.g. in Cellic and Celluclast bear quite different behaviour regarding the solid-liquid distribution, stability and cellulose and lignin affinity.

Comparing cost-optimal and net-zero energy targets in building retrofit

The recast of the European Unions Energy Performance of Buildings Directive introduced the concept of nearly zero-energy buildings, requiring its adoption from 2021 by all new buildings and existing ones submitted to major renovations. European Union member states must also ensure minimum energy requirements for buildings in order to achieve cost-optimal levels during their life cycle. Cost optimality and nearly zero-energy buildings are important concepts in European Union energy policies. These concepts are related, but one is more focused on costs, while the other is more concerned with low energy consumption and on site-renewable energy harvesting. If these approaches result in major differences in the selection of the best package of retrofit measures, then the transition from the cost-optimal concept to nearly zero-energy buildings might be problematic. To assess the most cost-effective solutions to achieve net-zero, a building in Porto, Portugal, was analysed. This determined not only the most cost-effective retrofit solutions but also compared these net-zero solutions with those resulting from the cost-optimal calculation. Both approaches lead to similar results, indicating that the transition between ‘cost optimality’ to ‘nearly zero-energy buildings’ could occur in Portugal.

Posttreatment of a brewery wastewater using a sequencing batch reactor

This study concerns the application of a sequencing batch reactor (SBR) for the posttreatment of an effluent rejected by an upflow anaerobic sludge blanket (UASB) reactor operating in a brewery. The goal was to achieve the required wastewater quality for discharge to surface water. The primary target was the removal of nitrogen compounds, but chemical oxygen demand and suspended solids were also concerns. Phosphorus concentration and protozoan population were also monitored during SBR operation. Two different strategies were tested: an operation based on an aerobic-anoxic sequence and another based on applying a predenitrification step, that is, an anoxic-aerobic-anoxic sequence. Ammonium (NH4-N) removal was achieved in all assays. Nitrification efficiency reached 97%, and the maximum observed rate was 0.175 kg NH4-N/kg volatile suspended solids.d. A denitrification process was detected during the aerated periods, despite a dissolved oxygen concentration in the bulk liquid of 2.8 to 3.7 mg O2/L. However, denitrification was suppressed when the bulk liquid oxygen concentration was increased to 7 mg O2/L. The carbon-to-nitrogen ratio of the UASB effluent was too low and hindered the postdenitrification phase. This fact was confirmed by complete nitrate removal when an acetate supplement was added. On the other hand, the insertion of a primary anoxic phase in the reaction cycle was the best treatment strategy, leading to nitrogen values within the legal framework. The protozoan population showed significant changes in response to the aerobic-anoxic conditions. However, periodic nonaerated conditions were not detrimental to aerobic protozoa, which recovered as soon as oxygen was again available.

Granulation during the start-up of a UASB reactor used in the treatment of low strength wastewaters

A glucose-based wastewater was efficiently degradaded by acidogenic bacteria, with a glucose removal efficiency close to 90%, and although a distinctive granular structure could not be observed, fluffy conglomerates developed in an Upflow Anaerobic Sludge Blanket (UASB) reactor. Subsequently, the pre-acidification of the wastewater promoted the granulation process. An enrichment in methanogenic bacteria was observed on the microscope and was confirmed by an increase in the specific methanogenic activity from 0.1 up to 0.5kgCOD/kgVSS.day. Such dynamics of microbial communities was also verified through changes in the polysaccharide and protein content, as well as in the electrophoretic mobility of the biomass.

Cellulase recycling in biorefineries—is it possible?

On a near future, bio-based economy will assume a key role in our lives. Lignocellulosic materials (e.g., agroforestry residues, industrial/solid wastes) represent a cheaper and environmentally friendly option to fossil fuels. Indeed, following suitable processing, they can be metabolized by different microorganisms to produce a wide range of compounds currently obtained by chemical synthesis. However, due to the recalcitrant nature of these materials, they cannot be directly used by microorganisms, the conversion of polysaccharides into simpler sugars being thus required. This conversion, which is usually undertaken enzymatically, represents a significant part on the final cost of the process. This fact has driven intense efforts on the reduction of the enzyme cost following different strategies. Here, we describe the fundamentals of the enzyme recycling technology, more specifically, cellulase recycling. We focus on the main strategies available for the recovery of both the liquid- and solid-bound enzyme fractions and discuss the relevant operational parameters (e.g., composition, temperature, additives, and pH). Although the efforts from the industry and enzyme suppliers are primarily oriented toward the development of enzyme cocktails able to quickly and effectively process biomass, it seems clear by now that enzyme recycling is technically possible.

Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw

The potential of enzymes recycling after hydrolysis and fermentation of wheat straw under a variety of conditions was investigated, monitoring the activity of the enzymes in the solid and liquid fractions, using low molecular weight substrates. A significant amount of active enzymes could be recovered by recycling the liquid phase. In the early stage of the process, enzyme adsorb to the substrate, then gradually returning to the solution as the saccharification proceeds. At 50°C, normally regarded as an acceptable operational temperature for saccharification, the enzymes (Celluclast) significantly undergo thermal deactivation. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Indeed, the amount of enzymes in the liquid phase increased with its thermostability and hydrolytic efficiency. This study contributes towards developing effective enzymes recycling strategies and helping to reduce the enzyme costs on bioethanol production.