Low-emissions system towards green fuels and high added-value bioactive compounds production based on Gordonia alkanivorans strain 1B biorefinery

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

Publication . 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.

2021-12Journal article

Open access

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

Publication . Pacheco, Marta; Paixão, Susana M.; Silva, Tiago; Alves, Luís

ABSTRACT: 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 mu moL g(-1) (DCW) h(-1) for Fru + Glu and from 1.91 to 3.58 mu moL g(-1) (DCW) h(-1) for JAJ, achieving a similar net 2-hydroxybiphenyl produced per g of consumed sugar (similar to 17 mu moL 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.

2019Journal article

Open access

A new biosurfactant/bioemulsifier from Gordonia alkanivorans Strain 1B: production and characterization

Publication . Silva, Tiago; Paixão, Susana M.; Tavares, João; Gil, Catia V.; Torres, Cristiana A. V.; Freitas, Filomena; Alves, Luís

ABSTRACT: 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.

2022-05Journal article

Open access

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

Publication . Tavares, João; Silva, Tiago; Paixão, Susana M.; Alves, Luís

ABSTRACT: 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.

2023-02Journal article

Embargoed access

A new impetus for biodesulfurization: bypassing sulfate inhibition in biocatalyst production

Publication . Silva, Tiago; Paixão, Susana M.; Alves, Luís

ABSTRACT: Biodesulfurization is a biotechnological process that employs microorganisms as biocatalysts to remove sulfur from fuels usually at mesophilic conditions, targeting recalcitrant organosulfur compounds without affecting their hydrocarbon structure. One of the bottlenecks hindering its large-scale application is the inhibition of biodesulfurization activity by easily metabolized sulfur compounds, such as sulfates, even at residual concentrations. This increases production costs by requiring high-purity sulfur-free nutrients or complex induction steps to prevent/revert inhibition. The objective of this work was to bypass this limitation and demonstrate that it is possible to produce biocatalysts with biodesulfurization activity using sulfate as the only sulfur source, without employing inducers or genetic manipulation, simply by adjusting the sulfur : carbon ratio in continuous culture. With this goal, the bacterium Gordonia alkanivorans strain 1B was cultivated in a chemostat with a medium containing 10 g L-1 of fructose as the carbon source and different sulfate concentrations (12-50 mg per L SO42-) using Na2SO4. Then the bacteria were employed as biocatalysts in biodesulfurization assays with a recalcitrant organosulfur compound (dibenzothiophene). Under these conditions it was observed that 2.2 mg(sulfate) g(fructose)(-1) ensured a biodesulfurization activity of 6.1 & mu;mol g(DCW)(-1) h(-1), 15% greater than previously reported for this strain with an inducer, without limiting biocatalyst production. This novel procedure was further applied to another biocatalyst, Rhodococcus erythropolis strain D1, validating its wide applicability to other desulfurizing microorganisms. Overall, these results indicate a previously unknown regulation mechanism dependent on relative sulfur concentration, which influences cellular responses and regulates biodesulfurization activity, allowing the use of easily metabolized sulfur sources to produce cost-effective biocatalysts for biodesulfurization.

2023-08Journal article

Open access