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- Biological upgrading of wastes from the pulp and paper industryPublication . Marques, Susana; Alves, Luís; Gírio, Francisco; Santos, J. A. L.; Roseiro, J. CarlosA process for biological upgrading of recycled paper sludge (RPS) was developed based on the enzymatic hydrolysis of major sludge components (cellulose and xylan) with commercial enzymes and fermentation of the resulting sugars into ethanol or lactic acid with adequate microbial strains. The process was implemented performing both steps sequentially (SHF) or simultaneously (SSF). Cellulosic and hemicellulosic fractions of RPS were completely converted by enzymatic hydrolysis (using Celluclast®1.5L with Novozym®188) into the constitutive glucose and xylose. Ethanol was produced from the RPS hydrolysate by the yeast Pichia stipitis CBS 5773. A slightly higher conversion yield was attained on SHF process, corresponding to an ethanol concentration of 19.6 g L-1, but 179 hours were needed. The SSF process was completed after 48 hours of incubation allowing the production of 18.6 g L-1 of ethanol from 178.6 g L-1 of dried RPS, corresponding to an overall conversion yield of 51% of the available carbohydrates on the initial substrate. Maximum production of lactic acid (LA) with Lactobacillus rhamnosus ATCC 7469 was obtained by performing the SSF process: 73 g L-1 of LA was achieved, corresponding to a maximum productivity of 2.9 g L-1 h-1, with 0.97 g LA produced per g of carbohydrates on initial sludge. The present results demonstrate the feasibility of the biological conversion of the ultimate waste obtained in the paper recycling loop into a biofuel (bioethanol) or an important chemical intermediate (LA, precursor of bioplastics), under the concept of a multi-purpose biorefinery.
- Influence of culture conditions towards optimal carotenoid production by Gordonia alkanivorans strain 1BPublication . Fernandes, Ana S.; Paixão, Susana M.; Silva, Tiago; Roseiro, J. Carlos; Alves, LuísABSTRACT: With the increasing awareness on the toxicity of several synthetic dyes, demand for pigments from natural sources, such as microbial carotenoids, has gained interest as a promising safe alternative colour additive. In this study, a surface response methodology based on the Doehlert distribution for two factors [% of glucose in a mixture of glucose + fructose (10 g/L total sugars), and sulfate concentration] was used towards the optimal carotenoids production by Gordonia alkanivorans strain 1B in the presence of light (400 lx). Time influence on pigment production by this bacterium was also evaluated, as well as the cell viability profile during longer incubation periods at optimal conditions. Indeed, the highest carotenoid production (2596-3100 mu g/g(DCW)) was obtained when strain 1B was cultivated in the optimal conditions: glucose 10 g/L and sulfate >= 22 mg/L, in the presence of light for 19 days at 30 degrees C, 150 rpm. Flow cytometry showed that the highest production was somehow related with the cellular stress. These results highlight the great potential of strain 1B as a new hyperpigment producer to be exploited towards several applications.
- Ionic liquids toward enhanced carotenoid extraction from bacterial biomassPublication . Silva, Tiago; Alves, Luís; Salgado, Francisco; Roseiro, J. Carlos; Lukasik, Rafal M.; Paixão, Susana M.ABSTRACT: Carotenoids are high added-value products primarily known for their intense coloration and high antioxidant activity. They can be extracted from a variety of natural sources, such as plants, animals, microalgae, yeasts, and bacteria. Gordonia alkanivorans strain 1B is a bacterium recognized as a hyper-pigment producer. However, due to its adaptations to its natural habitat, hydrocarbon-contaminated soils, strain 1B is resistant to different organic solvents, making carotenoid extraction through conventional methods more laborious and inefficient. Ionic liquids (ILs) have been abundantly shown to increase carotenoid extraction in plants, microalgae, and yeast; however, there is limited information regarding bacterial carotenoid extraction, especially for the Gordonia genus. Therefore, the main goal of this study was to evaluate the potential of ILs to mediate bacterial carotenoid extraction and develop a method to achieve higher yields with fewer pre-processing steps. In this context, an initial screening was performed with biomass of strain 1B and nineteen different ILs in various conditions, revealing that tributyl(ethyl)phosphonium diethyl phosphate (IL#18), combined with ethyl acetate (EAc) as a co-solvent, presented the highest level of carotenoid extraction. Afterward, to better understand the process and optimize the extraction results, two experimental designs were performed, varying the amounts of IL#18 and EAc used. These allowed the establishment of 50 µL of IL#18 with 1125 µL of EAc, for 400 µL of biomass (cell suspension with about 36 g/L), as the ideal conditions to achieve maximal carotenoid extraction. Compared to the conventional extraction method using DMSO, this novel procedure eliminates the need for biomass drying, reduces extraction temperatures from 50 °C to 22 ± 2 °C, and increases carotenoid extraction by 264%, allowing a near-complete recovery of carotenoids contained in the biomass. These results highlight the great potential of ILs for bacterial carotenoid extraction, increasing the process efficiency, while potentially reducing energy consumption, related costs, and emissions.
- Optimization of low sulfur jerusalem artichoke juice for fossil fuels biodesulfurization processPublication . Silva, Tiago; Paixão, Susana M.; Roseiro, J. Carlos; Alves, LuísMost of the world’s energy is generated from the burning of fossil fuels such as oil and its derivatives. When burnt, these fuels release into the atmosphere volatile organic compounds, sulfur as sulfur dioxide (SO2) and the fine particulate matter of metal sulfates. These are pollutants which can be responsible for bronchial irritation, asthma attacks, cardio-pulmonary diseases and lung cancer mortality, and they also contribute for the occurrence of acid rains and the increase of the hole in the ozone layer. For these reasons countries around the world imposed legal maxima to sulfur concentration on fuels. Forcing companies to develop methods of removing the sulfur contained in the oil. The most common is hydrodesulfurization which employs high pressures and temperatures associated with complex metal catalysts making it extremely expensive. So, it becomes important to explore alternatives such as biodesulfurization (BDS). This process is based on the use of microorganisms for the removal of sulfur form even from the most recalcitrant compounds at atmospheric pressure and temperature, making it cheaper and more eco-friendly. However it still presents some drawbacks, such as being easily inhibited in the presence of sulfates, which have been shown to have great inhibitory effect even in amounts as low as 6 mg/l [1]. In order to further reduce the costs associated with BDS it is possible to explore alternative carbon sources, as previously shown with carob pulp syrup and recycled paper sludge [1, 2]. The main objective of this work is the optimization of sulfate removal, from Jerusalem artichoke juice, in order to use it as an alternative carbon source for BDS.
- Optimization of a biphasic biodesulfurization systemPublication . Silva, Tiago; Paixão, Susana M.; Roseiro, J. Carlos; Alves, LuísABSTRACT: Many of the new generation fuels, although more sustainable, share some of the problems inherent to fossil fuels. Depending on the biomass/material that originated them, they can present different contaminants that can lead to environmental problems. Sulfur is one of the most common and problematic contaminants in fuels. It is released into the atmosphere in the form of SOx, leading to the formation of acid rains, which cause drastic environmental and infrastructural problems, as well as several types of health issues. High sulfur concentrations in fuels also result in a loss of efficiency of motors and energy generation systems, mostly due to corrosion and catalyst poisoning. The current thermochemical desulfurization process, hydrodesulfurization (HDS), is energy demanding, pollutant and has low efficiency against more complex organosulfur molecules. This led researchers to look for new alternatives. Biodesulfurization (BDS), is, as the name implies, the biological removal of sulfur from fuels using microorganisms as living biocatalysts. If correctly employed this process could be more efficient and less pollutant, since microorganisms directly target the sulfur atoms, even those present in complex molecular structures, such as dibenzothiophene (DBT). Moreover, microbial activity occurs at much lower temperatures and pressures, without the need for metal catalysts, resulting in a lower energy demand. While BDS is a promising technology, it is still at a low development stage, mostly due to some bottlenecks, which have been hindering its large-scale application. Similarly, to other biotechnological processes, it presents lower reaction rates, when compared to HDS, since it depends on the use of living organisms as catalysts. Furthermore, it must be performed under conditions that allow the microorganisms to maintain biological activity, limiting the range of applications. These conditions vary greatly depending on the microorganism selected, and their optimization can significantly increase the biodesulfurization activity of a biocatalyst.
- Optimization of low sulfur carob pulp liquor as carbon source for fossil fuels biodesulfurizationPublication . Silva, Tiago; Paixão, Susana M.; Teixeira, A. V.; Roseiro, J. Carlos; Alves, LuísBackground:Biodesulfurization (BDS) is a complementary technology to hydrodesulfurization since it allows the removal of recalcitrant sulfur compounds present in fossil fuels. The cost of culture medium to produce the biocatalysts is still one limitation for BDS application. Carob pulp, as an alternative carbon source, can reduce this cost. However, the presence of sulfates is critical, since BDS is inhibited at very low concentrations. Thus, the goal of this work was to optimize the process of sulfur precipitation on carob pulp liquor. Result:The effect of BaCl2 concentration (0–0.5%) and exposure time (6–36 h) on sulfate removal from carob pulp liquor was studied according to a statistical design following the Doehlert distribution for two factors. This experimental design determined that 0.5% BaCl2 concentration for 21 h were adequate conditions for sulfate removal from carob pulp liquor using BDS.Conclusion:These results demonstrate that it is possible to use alternative carbon sources derived from agro-industrial wastes for BDS, even those with high sulfur levels. For future industrial application, an inexpensive culture medium would have to be employed in a large-scale process and carob pulp liquor could be the carbon source.
- Jerusalem artichoke as low-cost fructose-rich feedstock for fossil fuels desulphurization by a fructophilic bacteriumPublication . Silva, Tiago; Paixão, Susana M.; Roseiro, J. Carlos; Alves, LuísAims: Through biodesulphurization (BDS) is possible to remove the sulphur present in fossil fuels to carry out the very strict legislation. However, this biological process is limited by the cost of the culture medium, and thus, it is important to explore cheaper alternative carbon sources, such as Jerusalem artichoke (JA). These carbon sources usually contain sulphates which interfere with the BDS process. The goal of this work was to remove the sulphates from Jerusalem artichoke juice (JAJ) through BaCl2 precipitation viewing the optimization of dibenzothiophene (DBT) desulphurization by Gordonia alkanivorans strain 1B. Methods and Results: Using a statistical design (Doehlert distribution), the effect of BaCl2 concentration (0·125–0·625%) and pH (5–9) was studied on sulphate concentration in hydrolysed JAJ. A validated surface response derived from data indicated that zero sulphates can be achieved with 0·5–0·55% (w/v) BaCl2 at pH 7; however, parallel BDS assays showed that the highest desulphurization was obtained with the juice treated with 0·5% (w/v) BaCl2 at pH 8·73. Further assays demonstrated that enhanced DBT desulphurization was achieved using hydrolysed JAJ treated in these optimal conditions. A total conversion of 400 µmol l-1 DBT into 2-hydroxybiphenyl (2-HBP) in <90 h was observed, attaining a 2-HBP maximum production rate of 28·2 µmol l-1 h-1 and a specific production rate of 5·06 µmol-1 g-1(DCW) h-1. Conclusions: These results highlight the efficacy of the treatment applied to JAJ in making this agromaterial a promising low-cost renewable feedstock for improved BDS by the fructophilic strain 1B. Significance and Impact of the Study: This study is a fundamental step viewing BDS application at the industrial level as it accounts a cost-effective production of the biocatalysts, one of the main drawbacks for BDS scale-up.
- Sugarcane bagasse delignification with potassium hydroxide for enhanced enzymatic hydrolysisPublication . Paixão, Susana M.; Ladeira, S. A.; Silva, Tiago; Arez, B. F.; Roseiro, J. Carlos; Martins, M. L. L.; Alves, LuísThe optimization of an alkaline pretreatment process for the delignification of sugarcane bagasse (SCB) to enhance the subsequent enzymatic hydrolysis was performed according to the Doehlert uniform shell design. In this experimental design, the effect of two factors—potassium hydroxide (KOH) concentration and autoclaving time at 121 C (1 atm)—on cellulose, hemicellulose, or the total polysaccharide and lignin content in SCB was evaluated. This response surface methodology revealed that KOH concentration is the factor that most influences the chemical characteristics of treated SCB (SCBt), with optimal conditions for the highest delignification being KOH in the range 5–10% (w/v) and an autoclaving time of 35 min, which provides an average of 97% total polysaccharides without inhibitor accumulation (furfural, 5-hydroxymethyl furfural) and #5% lignin. SCBt samples from two pretreatment conditions (KOH 3.25% – 13 min; KOH 10% – 35 min) were selected, based on the greatest delignification (70–74%) and polysaccharide availability (95–97%) after pretreatment, and further hydrolysed for fermentable sugar production. High sugar yields were obtained from both the pretreated samples (866 to 880 mg sugar per g biomass, respectively) in contrast with the 129 mg sugar per g raw biomass obtained from untreated SCB. These results demonstrate the effectiveness of KOH alkali pretreatments, which improves the overall digestibility of raw SCB polysaccharides from about 18% up to 91%. However, harsh alkali treatment (KOH 10%) is the most effective if the highest glucose/xylose ratio in the final sugar-rich hydrolysate is the aim. Hence, the use of sugar-rich hydrolysates obtained from SCBt as the carbon source for industrial purposes may provide a sustainable and economic solution for the production of bio-based added-value products, such as second generation (2G) bioethanol.
- KOH for enhanced sugarcane bagasse delignification and further production of sugar-rich hydrolyzates by enzymes applicationPublication . Paixão, Susana M.; Ladeira, S. A.; Arez, B. F.; Martins, M. L. L.; Roseiro, J. Carlos; Alves, LuísLignocellulosic biomass is envisaged as an important raw material for bioethanol production due to its low cost and high availability. Sugarcane bagasse (SCB), a fibrous residue of cane stalks left over after crushing and extraction of the juice from sugarcane; it is one of the largest cellulosic agro-industrial by-products. Tons of SCB are produced in Brazil as a waste of sugar and ethanol industries. This lignocellulosic by-product is a potential renewable source for 2G-bioethanol production. Usually, SCB is pretreated using alkaline and/or acid treatments viewing higher ethanol yields. The main goal of this study was to optimize the delignification of SCB towards the higher availability of glucans and xylans for further enzymatic hydrolysis to obtain sugar-rich syrups that will be more readily fermented to bioethanol. The delignification was carried out by autoclaving the biomass with KOH and the influence of KOH concentration (1-10%) and the autoclave time (10-60 min) were evaluated through a statistical design. Experimental distribution for two factors according to the Doehlert uniform design was used to produce response surfaces. The responses studied in this design were the percentage of hemicellulose, lignin and total polysaccharides. The results showed that from the two factors evaluated, the KOH concentration was the one that most influenced the response observed and that the treatments of SCB with KOH 5-10% for 35 minutes of autoclave at 121ºC and 1 atm led to the highest rates of lignin extraction. Using KOH treatment, a significant reduction of lignin content in SCB was observed, namely from 19% to 5%. Scanning electron micrographs of SCB pre-treated with 10% KOH for 35 minutes demonstrated a change in the structure of the material, with the appearance of broken structures, which can be attributed to the alkaline treatment. To validate the experiments, the SCB pretreated in the optimal conditions (95% of total polysaccharides) was further hydrolyzed with commercial enzymes and the enzymatic hydrolysis performance was evaluated.
- Application of ionic liquids for bacterial carotenoid extractionPublication . Salgado, Francisco; Silva, Tiago; Alves, Luís; Roseiro, J. Carlos; Lukasik, Rafal M.; Paixão, Susana M.ABSTRACT: One of the ways to make microbial bioprocesses more economically viable is to enhance valorization of high added value products resulting from the biomass, like carotenoids, which have a high market value. To recover these pigments from microbial biomass a good extraction method is required. Solvent extraction is one of the methods commonly used to extract carotenoids, however, solvent extractions are both material and time-consuming, and moreover also present some health and safety concerns. Ionic liquids (ILs) are a promising step forward to tackle some of these problems, even with their high price, and has been tested for the extraction of microorganism’s components. These “molten salts” are a group of compounds that have been known for a long time, but only in the last decades they have been attracting more attention from both researchers and industry. ILs are solvents that have a high solvation power for a wide range of molecules. ILs are salts with a melting point below 100ºC, which possess unique properties that depend on both the cation and anion present, high thermal and chemical stability, a large electrochemical window, great solvent power, non-flammability, and a negligible vapor pressure. Their versatility is one of their most attractive features, making them adaptable to many technologies. Therefore, ILs can be used to facilitate chemical reactions, extraction and separation, biotransformation, and can be used in biorefineries and other processes. As shown in previous works, Gordonia alkanivorans strain 1B has the capacity to produce carotenoids, however, since it was originally isolated form hydrocarbon rich environments, it is highly resistant to different organic solvents commonly used in extraction protocols. This makes the process slow and laborious, lowering yields and increasing solvent spending. As such, new extraction protocols must be developed and tested to obtain higher pigments yield. So, herein, the potential of ILs for carotenoids extraction was evaluated, since these compounds have been described as a good option to extract pigments produced by microorganisms.