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- A new biosurfactant/bioemulsifier from Gordonia alkanivorans Strain 1B: production and characterizationPublication . Silva, Tiago; Paixão, Susana M.; Tavares, João; Gil, Catia V.; Torres, Cristiana A. V.; Freitas, Filomena; Alves, LuísABSTRACT: Biosurfactants and bioemulsifiers (BS/BE) are naturally synthesized molecules, which can be used as alternatives to traditional detergents. These molecules are commonly produced by microorganisms isolated from hydrocarbon-rich environments. Gordonia alkanivorans strain 1B was originally found in such an environment, however little was known about its abilities as a BS/BE producer. The goal of this work was to access the potential of strain 1B as a BS/BE producer and perform the initial characterization of the produced compounds. It was demonstrated that strain 1B was able to synthesize lipoglycoprotein compounds with BS/BE properties, both extracellularly and adhered to the cells, without the need for a hydrophobic inducer, producing emulsion in several different hydrophobic phases. Using a crude BS/BE powder, the critical micelle concentration was determined (CMC = 16.94 mg/L), and its capacity to reduce the surface tension to a minimum of 35.63 mN/m was demonstrated, surpassing many commercial surfactants. Moreover, after dialysis, emulsification assays revealed an activity similar to that of Triton X-100 in almond and sunflower oils. In benzene, the E-24 value attained was 83.45%, which is 30% greater than that of the commercial alternative. The results obtained highlight for the presence of promising novel BS/BE produced by strain 1B.
- Development of a bench-scale photobioreactor with a novel recirculation system for continuous cultivation of microalgaePublication . Tavares, João; Silva, Tiago; Paixão, Susana M.; Alves, LuísABSTRACT: Microalgae cultivation can be used to increase the sustainability of carbon emitting processes, converting the CO2 from exhaust gases into fuels, food and chemicals. Many of the carbon emitting industries operate in a continuous manner, for periods that can span days or months, resulting in a continuous stream of gas emissions. Biogenic CO2 from industrial microbiological processes is one example, since in many cases it becomes unsustainable to stop these processes on a daily or weekly basis. To correctly sequester these emissions, microalgae systems must be operated under continuous constant conditions, requiring photobioreactors (PBRs) that can act as chemostats for long periods of time. However, in order to optimize culture parameters or study metabolic responses, bench-scale setups are necessary. Currently there is a lack of studies and design alternatives using chemostat, since most works focus on batch assays or semi-continuous cultures. Therefore, this work focused on the development of a continuous bench-scale PBR, which combines a retention vessel, a photocollector and a degasser, with an innovative recirculation system, that allows it to operate as an autotrophic chemostat, to study carbon sequestration from a biogenic CO2-rich constant air stream. To assess its applicability, the PBR was used to cultivate the green microalga Haematococcus pluvialis using as sole carbon source the CO2 produced by a coupled heterotrophic bacterial chemostat. An air stream containing ≈0.35 vol% of CO2, was fed to the system, and it was evaluated in terms of stability, carbon fixation and biomass productivity, for dilution rates ranging from 0.1 to 0.5 d−1. The PBR was able to operate under chemostat conditions for more than 100 days, producing a stable culture that generated proportional responses to the stimuli it was subjected to, attaining a maximum biomass productivity of 183 mg/L/d with a carbon fixation efficiency of ≈39% at 0.3 d−1. These results reinforce the effectiveness of the developed PBR system, making it suitable for laboratory-scale studies of continuous photoautotrophic microalgae cultivation.
- Hydrothermal alkaline sulfite pretreatment in the delivery of fermentable sugars from sugarcane bagassePublication . Tavares, João; Lukasik, Rafal M.; Paiva, Teresa de; Silva, Flávio daABSTRACT: This work aims to demonstrate the importance of hydrothermal alkaline sulfite pretreatment of sugarcane bagasse in the delivery of fermentable sugars. The pretreatment conditions, namely sodium sulfite loads (5.0-10.0 wt%), temperatures (140-160 degrees C) and reaction times (30-60 min), were tested according to the full factorial design of experiments. The employed pretreatment was characterized by high lignin removal (43-77 wt%) and partial extraction of xylans (up to 25.0 wt%), mainly by cleavage of acetyl and arabinosyl groups. After 72 h of enzymatic hydrolysis with 10 FPU (filter paper unit) cellulase and 10 CBU (cellobiose unit) -glucosidase per g of pretreated bagasse, the conversion of the carbohydrate-enriched substrates was in the range of 51 to 80 mol% for cellulose and 45 to 71 mol% for xylans. The best hydrothermal alkaline sulfite pretreatment conditions (10.0 wt% sulfite load, 150 degrees C and 45 min) were found to be highly efficient for the delivery of fermentable sugars with a yield as high as 40.4 +/- 1.9 g for 100 g native bagasse.
- Método para a determinação da atividade emulsificante de compostos surfactantes e/ou emulsificantes - Patente nacional: PT 117456 (B)Publication . Alves, Luís; Paixão, Susana M.; Tavares, João; Silva, TiagoRESUMO: A presente invenção traduz-se num método expedito para detetar e/ou quantificar a atividade emulsificante de compostos com propriedades surfactantes ou emulsificantes, a saber, detergentes domésticos e industriais, surfactantes sintéticos de uso laboratorial e biosurfactantes e/ou bioemulsificantes, presentes em soluções aquosas. Especificamente, o método compreende a definição de uma nova unidade de emulsificação, sendo esta o volume mínimo de uma solução do composto a testar, que diluído num mililitro de solução aquosa, é capaz de completa emulsificação de um mililitro de uma fase orgânica hidrofóbica.
- Desenvolvimento de um fotobiorreator, em escala de bancada, com um novo sistema de recirculação para cultivo contínuo de microalgas - Patente nacional: PT 118320 (A)Publication . Tavares, João; Alves, Luís; Paixão, Susana M.; Silva, TiagoRESUMO: A presente invenção traduz-se no Desenvolvimento de um fotobiorreactor (FBR) para o crescimento fotoautotrófico contínuo de microalgas, num sistema quimiostato, à escala de bancada. este novo fbr integra um vaso de retenção, que garante a mistura das fases líquidas e gasosas e centraliza o controle dos parâmetros e condições de cultura, um fotocoletor, que maximiza a área iluminada e a fixação do carbono através da fotossíntese, um desgaseificador, que previne a acumulação do oxigénio e assegura a recolha, e um novo conceito de recirculação que propicia uma cultura homogénea ao longo de todo o conjunto integrado funcionando como um único quimiostato. o protótipo do fbr desenvolvido está ilustrado no esquema da figura 1 (desenho sem considerar qualquer escala):(1) Recipiente de meio de cultura fresco; (2) Compressor de ar: g) fluxo de Ar (corrente gasosa contendo dióxido de carbono); (3) Bomba peristáltica; (4) Rotâmetro de precisão; (5) Bomba de recirculação da cultura autotrófica; (6) Câmara de gotejamento; (7) Conector em Y; (8) controlador de PH; (9) Banho termostatizado; (10) Vaso de retenção: A) Sensor de temperatura, B) Sonda de PH, C) Hélice de agitação magnética, D) Entrada da solução controladora de PH, E) Tubo borbulhador, F) Tubo nivelador; (11) Controlador de agitação; (12) Fotocoletor ¿ Série de colunas de bolhas (K); (13) Tomadas/ligações de acoplamento; (J) Cabeça lavadora de gases; (14) Desgaseificador: I) Tubo de descarga para colheita (para um recipiente de amostragem ou de colheita); (15) Condensador: H) Fluxo de ar de escape; (16) Analisador de gases.
- Design and validation of an expeditious analytical method to quantify the emulsifying activity during biosurfactants/bioemulsifiers productionPublication . Tavares, João; Alves, Luís; Silva, Tiago; Paixão, Susana M.ABSTRACT: Biosurfactants (BS) and bioemulsifiers (BE) are amphiphilic molecules that are produced by a wide range of microorganisms. Although the chemical composition of BS and BE is different, both BS/BE have recognized emulsifying properties, which are the focus of this study. Herein, a rapid and simple analytical method to quantify the emulsifying activity (EA) of a product produced by the actinomycete Gordonia alkanivorans strain 1B (BS/BE), which exhibits emulsifying properties, was developed. The analytical approach was based on the ability of a BS/BE solution to form a stable emulsion when mixed with n-heptane. So, using 4 mL screw cap glass tubes (10 x 75 mm, ND10 caps with PTFE septum), the EA was assessed by adding 1 mL of n-heptane to 1 mL of an aqueous solution containing the test product, mix by vortexing at high speed (2 min) and place the tube in an upright stable position for 10(min) before analyzing. A set of emulsification tests with increasing volumes of test product solutions was carried out until 100% emulsion was obtained in the organic phase. One emulsification unit was defined as the minimum volume of product (Vol(min) of emulsifier/surfactant, up to 1 mL) needed to form and maintain 100% emulsion in the organic phase. The corresponding emulsifying activity value is presented in U/mL, and it is calculated as: EA (product) = 1 U/Vol(min) (mL). Further validation by testing several synthetic surfactants and industrial/domestic dishwashing detergents, in parallel with the bacterial crude BS/BE, towards emulsifying activity determination (U/mL) was performed demonstrating the wide range of the method appli-cability. Moreover, the specific emulsifying activity for each product tested was estimated though correlation analysis (linear regression) between volumetric emulsifying activity (U/mL) and product concentration (g/L). Indeed, this new analytical approach to quantify the emulsifying activity is accurate and reproducible, and consequently it can be a promising tool to apply in screening/monitorization studies on BS/BE production enabling reliable comparisons.
- A fast and effective analytical method to quantify the emulsifying activity: design and validationPublication . Alves, Luís; Tavares, João; Silva, Tiago; Paixão, Susana M.ABSTRACT: Biosurfactants (BS) and bioemulsifiers (BE) are amphiphilic molecules that are produced by a wide range of microorganisms. According to Willumsen and Karlson, BS/BE are both surface active biomolecules, but while the surfactants play the role of surface tension reduction, emulsifiers are involved in formation and stabilization of emulsions. However, some biomolecules possess both surfactant and emulsifying properties, which contributes to their unique features, including high biodegradability, low toxicity, effectiveness at extremes of temperature, pH and salinity, and special biological activities (e.g., antimicrobial, antiviral, anticancer, etc). These attributes make them an alternative to their chemical counterparts and allows them to have key roles in several fields. In fact, the chemical composition of BS and BE is different, and this may contribute to their specific roles in nature and biotechnological applications; however, both BS/BE have recognized emulsifying properties, which are the focus of this study. The idea of quantifying the emulsifying activity rather than quantifying the emulsifiers and/or surfactants themselves has been studied before. The concept that stands out is the emulsification index (E24: % emulsification after 24 h) proposed by Cooper and Goldenberg [4], which is still applied, and some of its adaptations such as the one proposed by Trebbau de Acevedo and McInerney. These authors have defined one unit of emulsifying activity as the amount of emulsifier that results in an emulsification (E24) of 20%. Although theoretically simple, these approaches require considerable sample volume, have a long wait (24 h) and are lengthy. Moreover, they can be subjective, since two substances might induce complete emulsion at 24 h, with one resulting in a much denser emulsion. This may indicate more BS/BE activity; however, it is not easily comparable, or demonstratable. Furthermore, due to nature of emulsions and the factors that influence them, small differences in test conditions, such as shape or size of the tubes, or nature of the hydrophobic layer, can generate drastic differences, which hinders reproducibility between authors.
- A novel bench-scale photobioreactor for continuous cultivation of microalgae [Poster]Publication . Silva, Tiago; Tavares, João; Paixão, Susana M.; Alves, LuísABSTRACT: There are different types of carbon intensive industries. While some operate with daily intervals, others must be maintained in continuous operation, sometimes for weeks or months. Processes that depend on microbiological activity are usually of the second category, resulting in the continuous production of CO2 during extensive periods of time. In order to help mitigate climate change, alternative methods of carbon capture into added-value products have been the focus of research. Autotrophic microalgae cultures can be employed to sequester the carbon present in these streams, generating new products, while increasing process sustainability. However, to sequester these emissions microalgal bioreactors must also function under continuous constant conditions, requiring photobioreactors (PBRs) that can act as chemostats for long periods of time. Moreover, there is currently a lack of studies and design alternatives using microalgal chemostats. Most works tend to focus on batch assays or semi-continuous processes, presenting different responses depending on the growth stage of the culture, or the time of day. The present work is centred on the development of a novel continuous bench-scale PBR. This system uses an innovative recirculation concept to combine three different units (retention vessel, photocollector and degasser) that operate as a single autotrophic chemostat, allowing the study of carbon sequestration from a biogenic CO2-rich constant air stream. The novel PBR was tested by cultivating the microalga Haematococcus pluvialis at different dilution rates (0.1-0.5 d-1), while using as sole carbon source an air stream containing ≈0.35 vol% of CO2 (produced by a coupled heterotrophic bacterial chemostat). The results obtained revealed that the system could operate as a chemostat, allowing the production of stable cultures with proportional responses to the changes in dilution rates for more than 3 months, reaching a maximum biomass productivity of 183 mg/L/d, with a carbon fixation efficiency of ≈39% at 0.3 d-1. This makes the PBR prototype a promising tool to study/optimise integrated heterotrophic and autotrophic continuous processes, or constant sequestration of stable CO2-rich streams, making it easier to gather data for future scale-up.
- A novel microbial biosurfactant/bioemulsifier: production and characterizationPublication . Silva, Tiago; Paixão, Susana M.; Tavares, João; Alves, LuísABSTRACT: Currently there is an immediate need for new and more sustainable production methods in most industries. The detergent industry, frequently associated with negative environmental impacts, is also in need of new alternatives, such as biosurfactants/bioemulsifiers (BS/BE). These are naturally synthetized compounds, classified as amphiphilic, for having both hydrophobic and hydrophilic properties. Their application results in a reduction of the surface tension between two immiscible phases, facilitating the mixture of different substances such as water and oil, or water and air (Tavares et al., 2021). BS/BE present several advantages over conventional detergents, they have lower toxicity and greater biodegradability, resulting in lower negative impacts to both consumers and ecosystems. Furthermore, these compounds present antibiotic, antiviral and antioxidant properties. They can be used in a broad range of pH, temperature and salinity and are effective at small concentrations. This makes them interesting for many other industries, such as food, cosmetics, pharmaceutical and chemical. BS/BE are commonly produced by microorganisms found in particular environments such as oil wells, hydrocarbon contaminated soils and solid waste lixiviates. These compounds facilitate the access to hydrophobic nutrient sources abundant in these environments, while also increasing the resistance of the microorganisms to such toxic environments. Gordonia alkanivorans strain 1B, is a bacterium with significant biotechnological potential, which was isolated from oil contaminated soils (Alves et al., 2005). It is mostly known for its biodesulfurizing properties, carotenoid production and broad catabolic range (Silva et al., 2016). The present work focuses on the potential of this strain to produce BS/BE compounds, initial purification and characterization.
- Application of In-House Xylanases as an Addition to a Commercial Cellulase Cocktail for the Sustainable Saccharification of Pretreated Blue Agave Bagasse Used for Bioethanol ProductionPublication . Montiel, Carmina; Hernández-Meléndez, Oscar; Marques, Susana; Gírio, Francisco; Tavares, João; Ontañon, Ornella; Campos, Eleonora; Bárzana, EduardoABSTRACT: The study involves the use of commercial cellulase Cellic CTec2 in combination with two inhouse xylanases, PxXyn10A (XynA), a recombinant purified enzyme from Paenibacillus xylanivorans A59, and a xylanase enzymatic extract from native Moesziomyces aphidis PYCC 5535T (MaPYCC 5535T), for the enzymatic hydrolysis of pretreated blue agave bagasse (BAB) at the high solids load of 20% (w/v). Three different combinations of cellulase and xylanases were evaluated. When Cellic® CTec2 was used at a dosage of 10 FPU/g oven-dried solids (ODS) supplemented with XynA or MaPYCC 5535T at an endo-xylanase dosage of 100 U/g ODS, increases in the xylose yield of 30% and 33%, respectively, were obtained. When applying in-house xylanases alone (at an endo-xylanase dosage of 100 U/g ODS), xylan in BAB was selectively hydrolyzed into xylose with 5% yield with MaPYCC 5535T, while no xylose was detected with XynA. Interestingly, a synergic effect of Cellic® CTec 2 with both xylanases was observed when using a low dosage of 1 FPU/g ODS (allowing for some liquefaction of the reaction mixture), promoting xylose and glucose release by either xylanase. A higher concentration of monomeric sugars was obtained with 10 FPU/g ODS of Cellic® Ctec 2 supplemented with 100 U/g ODS of MaPYCC 5535T, followed by XynA. The improvement in saccharification through the synergistic combination of in-house xylanases and commercial cellulases allows for the obtention of sugar-rich hydrolysates, which enhances the technical sustainability of the process. Hydrolysates were then fermented using recombinant Cellux 4TM yeast to yield 45 g/L ethanol, representing an increase of about 30% with respect to the control obtained with only the commercial cellulase cocktail. The surface modification of agave biomass with the different combinations of enzymes was evidenced by scanning electron microscopy (SEM).