Streamlining the biodesulfurization process: development of an integrated continuous system prototype using Gordonia alkanivorans strain 1B.

Biodesulfurization is a biotechnological process that uses microorganisms as biocatalysts to actively remove sulfur from fuels. It has the potential to be cleaner and more efficient than the current industrial process, however several bottlenecks have prevented its implementation. Additionally, most works propose models based on direct cultivation on fuel, or batch production of biocatalysts followed by a processing step before application to batch biodesulfurization, which are difficult to replicate at a larger scale. Thus, there is a need for a model that can be adapted to a refining process, where fuel is being continuously produced to meet consumer needs. The main goal of this work was to develop the first bench-scale continuous biodesulfurization system that integrates biocatalyst production, biodesulfurization and fuel separation, into a single continuous process, taking advantage of the method for the continuous production of the biodesulfurization biocatalysts previously established. This system eliminates the need to process the biocatalysts and facilitates fuel separation, while mitigating some of the process bottlenecks. First, using the bacterium Gordonia alkanivorans strain 1B, continuous culture conditions were optimized to double biocatalyst production, and the produced biocatalysts were applied in batch biphasic biodesulfurization assays for a better understanding of the influence of different factors. Then, the novel integrated system was developed and evaluated using a model fuel (n-heptane + dibenzothiophene) in continuous biodesulfurization assays. With this system strain 1B surpassed its highest biodesulfurization rate, reaching 21 µmol h-1 g-1. Furthermore, by testing a recalcitrant model fuel, composed of n-heptane with dibenzothiophene and three alkylated derivatives (with 109 ppm of sulfur), 72% biodesulfurization was achieved by repeatedly passing the same fuel through the system, maintaining a constant response throughout sequential biodesulfurization cycles. Lastly, the system was also tested with real fuels (used tire/plastic pyrolysis oil; sweet and sour crude oils), revealing increased desulfurization activity. These results highlight the potential of the continuous biodesulfurization system to accelerate the transition from bench to commercial scale, contributing to the development of biodesulfurization biorefineries, centered on the valorization of sulfur-rich residues/biomasses for energy production.

Development of a bench-scale photobioreactor with a novel recirculation system for continuous cultivation of microalgae.

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.

Design and validation of an expeditious analytical method to quantify the emulsifying activity during biosurfactants/bioemulsifiers production.

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 × 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 (Volmin 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/Volmin (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 applicability. 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.

Effect of dibenzothiophene and its alkylated derivatives on coupled desulfurization and carotenoid production by Gordonia alkanivorans strain 1B.

Nowadays, the production of green transportation fuels is essential for a healthy life and environment. Effective and complete removal of organosulfur recalcitrant compounds from fuel oils is crucial to meet the stringent requirements of sulfur standards. However, the industry's solution (Hydrodesulfurization, HDS) is not effective in the removal of complex sulfur heterocyclic hydrocarbons. Thus, the development of more efficient and ecofriendly/sustainable desulfurization methods is critical, as either an alternative or a complement to HDS, foreseeing the production of ultra-low sulfur fuels (ULSF). Among the desulfurization techniques available, microbial desulfurization of organosulfur hydrocarbons (biodesulfurization, BDS) is attracting great attention. BDS is carried out at mild operation conditions, making it energetically cheaper and more ecofriendly, since it does not require hydrogen and produces far less greenhouse gases emission than HDS. In this context, the behavior of Gordonia alkanivorans strain 1B, a desulfurizing bacterium and hyper-pigment producer, was evaluated in the presence of four sulfur sources common in fuel oils: dibenzothiophene (DBT); 4-mDBT; 4,6-dmDBT and 4,6-deDBT (single/mixed), in terms of both desulfurization rate and overall carotenoid production. Simultaneously, the influence of the carbon source used (fructose vs glucose) on the overall effectiveness of the coupled bioprocesses was also assessed. The results obtained highlight the potential of strain 1B to desulfurize all the tested recalcitrant compounds and simultaneously produce carotenoids. However, the highest BDS values were observed for 4,6-deDBT (5.75 µmol/g (DCW)/h) and for the mix of DBTs (5.20 µmol/g (DCW)/h), when fructose was used as carbon source. Indeed, when the mixture of DBTs ("model oil surrogate") was desulfurized by cells growing in fructose both desulfurization rate and total pigments amount were higher than those observed for glucose growing cells. Moreover, under these conditions, the strain 1B was able to produce high added-value carotenoids, namely astaxanthin, lutein and canthaxanthin. Hence, these results are promising when aiming to achieve a scale-up scenario. In fact, the inclusion of the production of high added-value products within a BDS process targeting ULSF may be a sustainable way to turn its scale-up economically viable.

Prognostic power of anaerobic threshold parameters in patients with transposition of the great arteries and systemic right ventricle.

INTRODUCTION: Both transposition of the great arteries (TGA) previously submitted to a Senning/Mustard procedure and congenitally corrected TGA (cc-TGA) have the systemic circulation supported by the morphological right ventricle, thereby rendering these patients to heart failure events risk. The aim of this study was to evaluate cardiopulmonary exercise test parameters for stratifying the risk of heart failure events in TGA patients. METHODS: Retrospective evaluation of adult TGA patients with systemic circulation supported by the morphological right ventricle submitted to cardiopulmonary exercise test in a tertiary centre. Patients were followed up for at least 1 year for the primary endpoint of cardiac death or heart failure hospitalisation. Several cardiopulmonary exercise test parameters were analysed as potential predictors of the combined endpoint and their predictive power were compared (area under the curve). RESULTS: Cardiopulmonary exercise test was performed in 44 TGA patients (8 cc-TGA), with a mean age of 35.1 ± 8.4 years. The primary endpoint was reached by 10 (22.7%) patients, with a mean follow-up of 36.7 ± 26.8 months. Heart rate at anaerobic threshold had the highest area under the curve value (0.864), followed by peak oxygen consumption (pVO2) (0.838). Heart rate at anaerobic threshold ≤95 bpm and pVO2 ≤20 ml/kg/min had a sensitivity of 87.5 and 80.0% and a specificity of 82.4 and 76.5%, respectively, for the primary outcome. CONCLUSION: Heart rate at anaerobic threshold ≤95 bpm had the highest predictive power of all cardiopulmonary exercise test parameters analysed for heart failure events in TGA patients with systemic circulation supported by the morphological right ventricle.

On the road to cost-effective fossil fuel desulfurization by Gordonia alkanivorans strain 1B.

Biodesulfurization (BDS) is an ecofriendly process that uses microorganisms to efficiently remove sulfur from fossil fuels. To make the BDS process economically competitive with the deep hydrodesulfurization process, which is currently used in the oil industry, it is necessary to improve several factors. One crucial limitation to be overcome, common within many other biotechnological processes, is the cost of the culture medium. Therefore, an important line of work to make BDS scale-up less costly is the optimization of the culture medium composition aiming to reduce operating expenses and maximize biocatalyst production. In this context, the main goal of this study was on the minimization of inorganic key components of sulfur-free mineral (SFM) medium in order to get the maximal production of efficient desulfurizing biocatalysts. Hence, a set of assays was carried out to develop an optimal culture medium containing minimal amounts of nitrogen (N) and magnesium (Mg) sources and trace elements solution (TES). These assays allowed the design of a SFMM (SFM minimum) medium containing 85% N-source, 25% Mg-source and 25% TES. Further validation consisted of testing this minimized medium using two carbon sources: the commercial C-source (glucose + fructose) versus Jerusalem artichoke juice (JAJ) as a cheaper alternative. SFMM medium allowed microbial cells to almost duplicate their specific desulfurization rate (q 2-HBP) for both tested C-sources, namely from 2.15 to 3.39 µmoL g-1 (DCW) h-1 for Fru + Glu and from 1.91 to 3.58 µmoL g-1 (DCW) h-1 for JAJ, achieving a similar net 2-hydroxybiphenyl produced per g of consumed sugar (∼17 µmoL g-1). These results point out the great advantage of using cheaper culture medium that in addition enhances the bioprocess effectiveness, paving the way to a sustainable scale-up for fossil fuel BDS.

Influence of culture conditions towards optimal carotenoid production by Gordonia alkanivorans strain 1B.

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 µg/gDCW) 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 °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.

Herb-Drug Interactions: An Insight into Cardiovascular Diseases Based on Case Reports.

Cardiovascular patients frequently use herbal medicinal products, in order to contribute to the improvement of their chronic condition without medical intervention. However, they are likely to suffer from adverse effects from natural products and herb-drug interactions. In this work we present the results collected from a public campaign "Learning Health, among Plants and Medicines", carried out by the Observatory of Herb-Drug Interactions (www.oipm.uc.pt), to alert cardiovascular patients and healthcare providers for the potential occurrence of herb-drug interactions with cardiovascular therapy. From the data received, it was highlighted the prevalence of certain natural products used by many cardiovascular patients in Portugal, particularly goji berries, green tea, mangosteen and rooibos that have significant cardiovascular effects. For this reason their intake should be carefully monitored in these patients. This prevalence of consumption suggests a pattern in their use in Portugal and a prevention of herb-drug interactions should be carried out by the health professionals. The ending results also indicate that there is still a lack of knowledge about the possible risks of herbal products intake, which may adversely affect the health of any patient. Thus becomes clear the value of the role of health professionals in the screening of such interactions.

Screening of novel yeast inulinases and further application to bioprocesses.

Inulin is a carbohydrate composed of linear chains of ß-2,1-linked D-fructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end. Jerusalem artichoke (JA) is one of the most interesting materials among unconventional and renewable raw materials, with levels of inulin reaching 50-80% of dry matter. Inulin or inulin-rich materials can be actively hydrolyzed by microbial inulinases to produce glucose and fructose syrups that can be used in bioprocesses. In this study, several microbial strains were isolated and their ability to inulinase biosynthesis was evaluated. The novel yeast strain Talf1, identified as Zygosaccharomyces bailii, was the best inulinase producer, attaining 8.67 U/ml of inulinase activity when JA juice was used as the inducer substrate. Z. bailii strain Talf1 and/or its enzymatic crude extract were further applied for bioethanol production and biodesulfurization (BDS) processes, using inulin and JA juice as carbon source. In a consolidated bioprocessing for ethanol production from 200 g/l inulin, Z. bailii strain Talf1 was able to produce 67 g/l of ethanol. This ethanol yield was improved in a simultaneous saccharification and fermentation (SSF) process, with the ethanologenic yeast Saccharomyces cerevisiae CCMI 885 and the Talf1 inulinases, achieving a production of 78 g/l ethanol. However, the highest ethanol yield (∼48%) was obtained in a SSF process from JA juice (∼130 g/l fermentable sugars), where the S. cerevisiae produced 63 g/l ethanol. Relatively to the dibenzothiophene BDS tests, the Gordonia alkanivorans strain 1B achieved a desulfurization rate of 4.8 µM/h within a SSF process using Talf1 inulinases and JA juice, highlighting the potential of JA as a less expensive alternative carbon source. These results showed the high potential of Z. bailii strain Talf1 inulinases as a versatile tool for bioprocesses using inulin-rich materials.

Phase synchronization in the perturbed Chua circuit.

We show experimental and numerical results of phase synchronization between the chaotic Chua circuit and a small sinusoidal perturbation. Experimental real-time phase synchronized states can be detected with oscilloscope visualization of the attractor, using specific sampling rates. Arnold tongues demonstrate robust phase synchronized states for perturbation frequencies close to the characteristic frequency of the unperturbed Chua.