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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.

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 (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 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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/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 °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.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)