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.

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.

A novel ß-xylosidase from Anoxybacillus sp. 3M towards an improved agro-industrial residues saccharification.

An intracellular ß-xylosidase (AbXyl), from the thermoalkaline Anoxybacillus sp. 3M, was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+ inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by ß-mercaptoethanol. In the presence of p-nitrophenyl-ß-d-xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s-1, Kcat Km-1 of 2322 s-1 mM and was moderately influenced by xylose (Ki 21.25 mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars.

Protein profiling as early detection biomarkers for TiO2 nanoparticle toxicity in Daphnia magna.

The mode of action for nanoparticle (NP) toxicity in aquatic organisms is not yet fully understood. In this work, a strategy other than toxicity testing was applied to Daphnia magna exposed to TiO2-NPs: the use of nuclear microscopy and the assessment of protein profile. D. magna is a keystone species broadly used as a model system in ecotoxicology. Titanium (Ti) was found in the D. magna digestive tract, mainly in the gut. The penetration of Ti into the epithelial region was greater at higher exposure levels and also observed in eggs in the brood pouch. The protein profile of individuals exposed to different concentrations showed that 2.8 and 5.6 mg/L TiO2-NP concentrations induced an over-expression of the majority of proteins, in particular proteins with molecular weight of ∼120, 85 and 15 kDa, while 11.2 mg/L TiO2-NP had an inhibitory effect on protein expression. The Matrix-assisted laser desorption ionization with tandem time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of these proteins consistently identified them as vitellogenin (Vtg)-like proteins, associated with enzymes involved in redox balance. These results indicate that Vtg-like proteins are up-regulated in D. magna exposed to TiO2-NPs. Vitellogenesis is associated with the reproduction system, suggesting that TiO2-NP exposure can impair reproduction by affecting this process. The precise mode of action of TiO2-NPs is still unclear and the results from this study are a first attempt to identify specific proteins as potential markers of TiO2-NP toxicity in D. magna, providing useful information for future research.

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.

High-pressure carbon dioxide/water pre-treatment of sugarcane bagasse and elephant grass: Assessment of the effect of biomass composition on process efficiency.

The performance of two lignocellulosic biomasses was studied in high-pressure carbon dioxide/water pre-treatment. Sugarcane bagasse and elephant grass were used to produce C5-sugars from hemicellulose and, simultaneously, to promote cellulose digestibility for enzymatic saccharification. Different pre-treatment conditions, with combined severity factor ranging from -1.17 to -0.04, were evaluated and maximal total xylan to xylose yields of 59.2wt.% (34.4wt.% xylooligomers) and 46.4wt.% (34.9wt.% xylooligomers) were attained for sugarcane bagasse and elephant grass, respectively. Furthermore, pre-treated biomasses were highly digestible, with glucan to glucose yields of 77.2mol% and 72.4mol% for sugarcane bagasse and elephant grass, respectively. High-pressure carbon dioxide/water pre-treatment provides high total C5-sugars and glucose recovery from both lignocellulosic biomasses; however it is highly influenced by composition and intrinsic features of each biomass. The obtained results confirm this approach as an effective and greener alternative to conventional pre-treatment processes.

Simultaneously saccharification and fermentation approach as a tool for enhanced fossil fuels biodesulfurization.

Biodesulfurization can be a complementary technology to the hydrodesulfurization, the commonly physical-chemical process used for sulfur removal from crude oil. The desulfurizing bacterium Gordonia alkanivorans strain 1B as a fructophilic microorganism requires fructose as C-source. In this context, the main goal of this work was the optimization of a simultaneous saccharification and fermentation (SSF) approach using the Zygosaccharomyces bailii strain Talf1 crude enzymes with invertase activity and sucrose as a cheaper fructose-rich commercial C-source (50% fructose) towards dibenzothiophene (DBT) desulfurization by strain 1B. The determination of optimal conditions, for both sucrose hydrolysis and DBT desulfurization was carried out through two sequential experimental uniform designs according to the Doehlert distribution for two factors: pH (5.5-7.5) and temperature (28-38 °C), with the enzyme load of 1.16 U/g/L; and enzyme load (0-4 U/g/L) and temperature (28-38 °C), with pH at 7.5. Based on 2-hydroxybiphenyl production, the analysis of the response surfaces obtained pointed out for pH 7.5, 32 °C and 1.8 U/g/L as optimal conditions. Further optimized SSF of sucrose during the DBT desulfurization process permitted to attain a 4-fold enhanced biodesulfurization. This study opens a new focus of research through the exploitation of sustainable low cost sucrose-rich feedstocks towards a more economical viable bioprocess scale-up.

Properties of an alkali-thermo stable xylanase from Geobacillus thermodenitrificans A333 and applicability in xylooligosaccharides generation.

An extracellular thermo-alkali-stable and cellulase-free xylanase from Geobacillus thermodenitrificans A333 was purified to homogeneity by ion exchange and size exclusion chromatography. Its molecular mass was 44 kDa as estimated in native and denaturing conditions by gel filtration and SDS-PAGE analysis, respectively. The xylanase (GtXyn) exhibited maximum activity at 70 °C and pH 7.5. It was stable over broad ranges of temperature and pH retaining 88 % of activity at 60 °C and up to 97 % in the pH range 7.5-10.0 after 24 h. Moreover, the enzyme was active up to 3.0 M sodium chloride concentration, exhibiting at that value 70 % residual activity after 1 h. The presence of other metal ions did not affect the activity with the sole exceptions of K(+) that showed a stimulating effect, and Fe(2+), Co(2+) and Hg(2+), which inhibited the enzyme. The xylanase was activated by non-ionic surfactants and was stable in organic solvents remaining fully active over 24 h of incubation in 40 % ethanol at 25 °C. Furthermore, the enzyme was resistant to most of the neutral and alkaline proteases tested. The enzyme was active only on xylan, showing no marked preference towards xylans from different origins. The hydrolysis of beechwood xylan and agriculture-based biomass materials yielded xylooligosaccharides with a polymerization degree ranging from 2 to 6 units and xylobiose and xylotriose as main products. These properties indicate G. thermodenitrificans A333 xylanase as a promising candidate for several biotechnological applications, such as xylooligosaccharides preparation.

Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization.

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml(-1)) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5-6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5-6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l(-1) sucrose and 250 µM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l(-1) sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its µmax from 0.035 to 0.070 h(-1) and attained a 2-hydroxybiphenyl productivity of 5.80 µM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.

Influence of the carbon source on Gordonia alkanivorans strain 1B resistance to 2-hydroxybiphenyl toxicity.

The viability of bacteria plays a critical role in the enhancement of fossil fuels biodesulfurization efficiency since cells are exposed to toxic compounds such as 2-hydroxybiphenyl (2-HBP), the end product of dibenzothiophene (DBT) biodesulfurization. The goal of this work was to study the influence of the carbon source on the resistance of Gordonia alkanivorans strain 1B to 2-HBP. The physiological response of this bacterium, pre-grown in glucose or fructose, to 2-HBP was evaluated using two approaches: a growth inhibition toxicity test and flow cytometry. The results obtained from the growth inhibition bioassays showed that the carbon source has an influence on the sensitivity of strain 1B growing cells to 2-HBP. The highest IC50 value was obtained for the assay using fructose as carbon source in both inoculum growth and test medium (IC50-48 h = 0.464 mM). Relatively to the evaluation of 2-HBP effect on the physiological state of resting cells by flow cytometry, the results showed that concentrations of 2-HBP >1 mM generated significant loss of cell viability. The higher the 2-HBP concentration, the higher the toxicity effect on cells and the faster the loss of cell viability. In overall, the flow cytometry results highlighted that strain 1B resting cells grown in glucose-SO4 or glucose-DBT are physiologically less resistant to 2-HBP than resting cells grown in fructose-SO4 or fructose-DBT, respectively.

Enhancement of dibenzothiophene desulfurization by Gordonia alkanivorans strain 1B using sugar beet molasses as alternative carbon source.

There are several problems limiting an industrial application of fossil fuel biodesulfurization, and one of them is the cost of culture media used to grow the microorganisms involved in the process. In this context, the utilization of alternative carbon sources resulting from agro-industrial by-products could be a strategy to reduce the investment in the operating expenses of a future industrial application. Recently, Gordonia alkanivorans 1B was described as a fructophilic desulfurizing bacterium, and this characteristic opens a new interest in alternative carbon sources rich in fructose. Thus, the goal of this study was to evaluate the utilization of sugar beet molasses (SBM) in the dibenzothiophene (DBT) desulfurization process using strain 1B. SBM firstly treated with 0.25% BaCl2 (w/v) was used after sucrose acidic hydrolysis or in a simultaneous saccharification and fermentation process with a Zygosaccharomyces bailii Talf1 invertase (1%), showing promising results. In optimal conditions, strain 1B presented a µ max of 0.0795 h(-1), and all DBT was converted to 2-hydroxybiphenyl (250 µM) within 48 h with a maximum production rate of 7.78 µM h(-1). Our results showed the high potential of SBM to be used in a future industrial fossil fuel biodesulfurization process using strain 1B.

Fructophilic behaviour of Gordonia alkanivorans strain 1B during dibenzothiophene desulfurization process.

Biodesulfurization (BDS) aims at the removal of recalcitrant sulfur from fossil fuels at mild operating conditions with the aid of microorganisms. These microorganisms can remove sulfur from dibenzothiphene (DBT), a model compound, or other polycyclic aromatic used as sulfur source, making BDS an easy and environmental friendly process. Gordonia alkanivorans strain 1B has been described as a desulfurizing bacterium, able to desulfurize DBT to 2-hydroxybiphenyl (2-HBP), the final product of the 4S pathway, using d-glucose as carbon source. However, both cell growth and desulfurization can be largely affected by the nutrient composition of the growth medium, due to cofactor requirements of many enzymes involved in the BDS biochemical pathway. In this study, the main goal was to investigate the influence of several sugars, as carbon source, on the growth and DBT desulfurization ability of G. alkanivorans strain 1B. The results of desulfurization tests showed that the lowest values for the growth rate (0.025 hour(-1)) and for the overall 2-HBP production rate (1.80 µm/hour) by the strain 1B were obtained in glucose grown cultures. When using sucrose, the growth rate increase exhibited by strain 1B led to a higher biomass productivity, which induced a slightly increase in the 2-HBP production rate (1.91 µm/hour), conversely in terms of 2-HBP specific production rate (q2-HBP) the value obtained was markedly lower (0.718 µmol/g/hour in sucrose versus 1.22 µmol/g/hour in glucose). When a mixture of glucose and fructose was used as carbon source, strain 1B reached a value of q2-HBP=1.90 µmol/g/hour, close to that in fructose (q2-HBP=2.12 µmol/g/hour). The highest values for both cell growth (µ=0.091 hour(-1)) and 2-HPB production (9.29µm/hour) were obtained when strain 1B was desulfurizing DBT in the presence of fructose as the only carbon source, indicating a fructophilic behaviour by this bacterium. This fact is in agreement with the highest value of biomass productivity by strain 1B be in fructose, which resulted in a higher amount cells fulfilling the DBT-desulfurization. The greater number of functional cells conducted to a more effectiveness BDS process by strain 1B, as they attained a q2-HBP about 74% higher than in glucose grown cultures. Moreover, this significant BDS enhancement can better be observed in terms of the overall 2-HBP production rate, which increased over 5-fold, from 1.80 µm/hour (in glucose) to 9.29 µm/hour (in fructose).

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.

Ecotoxicological assessment of industrial wastewaters in Trancão River Basin (Portugal).

It is important to assess the toxicity of complex effluents, since chemical evaluation alone is insufficient to protect the environment. Direct Toxicity Assessment is valuable in the decision process regarding the final disposal of complex wastewaters as it measures the total effects of the discharge, because of its known and unknown chemicals, additionally having some degree of ecological relevance. In Portugal, ecotoxicity tests are not used on a regular basis to control wastewaters. So, an integrated ecotoxicological, physical, and chemical study of wastewaters from 17 industries, in the Trancão River Basin, was carried out viewing proposing a test battery to be used in wastewater evaluation. An approach which does not include an ecotoxicological characterization may not properly evaluate the potential risks of effluent discharges, especially when they are complex. From the study carried out the use of a battery of assays to apply in the monitoring of complex wastewaters was proposed, including Microtox test, Daphnia test, and an algal test. Moreover, the added value of the ecotoxicological assessment of industrial wastewaters was demonstrated and could support the implementation of EU Directives (e.g. IPPC, WFD) within the Portuguese situation.

Performance of a miniaturized algal bioassay in phytotoxicity screening.

A miniaturized and low-cost algal growth-inhibition assay, with Pseudokirchneriella subcapitata, based on the standard ISO 8692 and using 96-well microplates, was tested and optimized in this work, to be used as a useful tool for pollutant phytotoxicity screening. For validation, the performance of the microplate algal growth-inhibition assay was first compared with the standard flask assay for the toxicity testing of five reference toxicants (copper(II) sulfate, zinc sulfate, potassium permanganate, potassium dichromate and 3,5-Dichlorophenol) and six wastewater samples. Statistical evaluation of EC(50) results from both methods demonstrated a good agreement between microplate and flask assays either in testing chemicals (r (2) = 0.975, p < 0.0017) or environmental samples toxicity (r (2) = 0.984, p < 0.0001). In addition, the performance of this algal microplate bioassay was also evaluated in comparison with Lemna test, ISO 20079, for phytotoxicity assessment of 27 wastewater samples from industries and treatment plants. The results showed that the algal test was more sensitive for most of the samples, but a significant agreement between both tests was observed (r (2) = 0.644, p < 0.0001). In conclusion, this miniaturized test can be a good tool to include in a battery of tests for phytotoxicity screening of a wide range of chemicals and environmental samples, with the advantage of requiring low sample volumes for the test, allowing large numbers of samples to be tested, and generating low volumes of waste.