Improved rhamnolipid biosurfactant production by Burkholderia thailandensis E264 using agro-industrial waste

Biosurfactants are amphiphilic surface-active compounds, produced by various microorganisms, that reduce surface and interfacial tension. These compounds are attracting increasing interest over their chemical counterparts due to their advantages, such as biodegradability, high stability in extreme environments, low toxicity, low critical micelle concentrations (CMC) and the fact that they can be effectively produced from agro-industrial wastes and renewable resources. Furthermore, their diversity allows for a significant number of uses, including microbial enhanced oil recovery (MEOR), bioremediation and biomedical applications. However, the high operational costs, mainly regarding the use of expensive raw materials in the fermentation and the complex downstream processing, to yield low amounts of product restricts their industrial-scale applications. Several attempts to solve these limitations by reducing the production costs have been conducted and include the use of low-cost agro-industrial wastes and by-products as substrates. One of these low-cost substrates, that has been successfully used to produce biosurfactants by Bacillus subtilis and Pseudomonas aeruginosa strains, is Corn Steep Liquor (CSL). In this research, rhamnolipid biosurfactant production by Burkholderia thailandensis E264 was optimized using this agro-industrial waste as sole substrate. When grown in a culture medium containing CSL (7.5% v/v), this strain produced 1.77 g biosurfactant/L, which is about 2.6 times the amount of biosurfactant produced in the standard synthetic medium. The purified biosurfactant produced in the low-cost medium exhibited similar surface-active properties when compared with that produced in the synthetic medium, reducing the surface tension of water to 29.7 mN/m, with a CMC of 385 mg/L. HPLC analysis showed that the culture medium used contains about 2.6 g/L of fructose and 2.5 g/L of glucose, that are fully consumed within the first 48 h of fermentation. Since the synthetic medium contains 40 g/L of glycerol, results suggest that biosurfactant production is more efficient in the low-cost medium. Furthermore, to the best of the authors knowledge, this is the first experimental research that combines the utilization of B. thailandensis with CSL to produce biosurfactants with very optimistic results in terms of cost and production levels. The rhamnolipid-containing cell-free supernatant could be used directly in bioremediation or MEOR processes.This study was supported by PARTEX Oil and Gas, and the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte 2020 - Programa Operacional Regional do Norte. The authors also acknowledge to the Biomass and Bioenergy Research Infrastructure (BBRI)- LISBOA-01-0145-FEDER-022059, supported by Operational Program for Competitiveness and Internationalization (PORTUGAL2020), by Lisbon Portugal Regional Operational Program (Lisboa 2020) and by North Portugal Regional Operational Program (Norte 2020) under the Portugal 2020 Partnership Agreement, through the ERDF.info:eu-repo/semantics/publishedVersio

Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?

Astaxanthin is a xanthophyll of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from β-carotene by the sequential action of two enzymes: a β-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3') positions of each of the two β-ionone rings of β-carotene, and a β-carotene ketolase that introduces keto groups at carbons 4 and 4' of the β-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of β-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementation studies of X. dendrorhous mutants and expression analysis in Mucor circinelloides reveals that the CrtS enzyme is a β-carotene hydroxylase of the P-450 monooxygenase family that converts β-carotene to the hydroxylated derivatives β-cryptoxanthin and zeaxanthin, but it does not form astaxanthin or the ketolated intermediates in this fungus. A bifunctional β-carotene hydroxylase-ketolase activity has been proposed for the CrtS protein. The evidence for and against this hypothesis is analyzed in detail in this review

Development of a low-cost culture medium for biopolymer production by Rhizobium viscosum CECT 908 and its potential application in Microbial Enhanced Oil Recovery

Polymers are a versatile class of compounds that play an essential role in our society, being their production estimated in more than 180 million tons per year. Nowadays, the world market is dominated by synthetic and plant-derived polymers. Biopolymers of microbial origin are characterized by their better environmental compatibility and biodegradability when compared with the synthetic ones, and their production is faster than those obtained from plants. Microbial biopolymers usually exhibit excellent rheological properties, stability at a wide range of temperatures, salinities and pH values, as well as a broad variety of chemical structures, which results in different physicochemical and rheological properties. However, despite their outstanding properties, their application is still limited by their high production costs. In this work, an alternative low-cost culture medium was developed for biopolymer production by Rhizobium viscosum CECT 908, containing sugarcane molasses (60 g/L) and corn steep liquor (1%, v/v) as carbon and nitrogen sources, respectively. Using this low-cost medium, higher biopolymer production and apparent viscosity values (5.2 g/L and 6700 mPa s, respectively) were obtained comparing with the synthetic medium (2.3 g/L and 1100 mPa s), which contained glucose and yeast extract. As a result, the cost of the culture medium necessary to produce 1 Kg of biopolymer was reduced more than 20 times. The biopolymer produced in the alternative low-cost medium exhibited better rheological properties as compared to xanthan gum, including higher viscosity at the same concentration. Furthermore, it was found to be stable at temperatures up to 80ºC, NaCl concentrations as high as 200 g/L, and high shear rates (300 s-1). Polymers are widely used by the oil industry to increase the oil reservoirs productivity during the tertiary oil recovery processes. In sand-pack column assays performed using a heavy crude oil (40ºC= 170 mPa s), this biopolymer produced using the low-cost medium demonstrated a better performance than xanthan gum, recovering almost 50% of the entrapped oil. Results herein obtained highlight that the R. viscosum biopolymer is a promising candidate for application in MEOR as an alternative to the conventional microbial and synthetic polymers currently used.info:eu-repo/semantics/publishedVersio

Improved rhamnolipid biosurfactant production by Burkholderia thailandensis E264 using agro-industrial wastes

Biosurfactants are amphiphilic surface-active compounds, produced by various microorganisms, that reduce surface and interfacial tension. These compounds are attracting increasing interest over their chemical counterparts due to their advantages, such as biodegradability, high stability in extreme environments, low toxicity, low critical micelle concentrations (CMC) and the fact that they can be effectively produced from agro-industrial wastes and renewable resources. Furthermore, their diversity allows for a significant number of uses, including microbial enhanced oil recovery (MEOR), bioremediation and biomedical applications. However, the high operational costs, mainly regarding the use of expensive raw materials in the fermentation and the complex downstream processing due to the low production yields restricts their industrial-scale applications. Several attempts to solve these limitations by reducing the production costs have been conducted and include the use of low-cost agro-industrial wastes and by-products as substrates. One of these low-cost substrates, that has been successfully used to produce biosurfactants by Bacillus subtilis and Pseudomonas aeruginosa, is corn steep liquor (CSL). In this work, CSL is evaluated for rhamnolipid biosurfactant production by Burkholderia thailandensis E264. When grown in a culture medium containing CSL (7.5% v/v) as sole substrate, this strain produced 1.8 g rhamnolipid/L, which is about 2.6 times the amount of rhamnolipid produced in the standard synthetic medium. By supplementing the culture medium with olive oil mill wastewater (OMW, 10% v/v), a residue originating from the olive oil extraction industry, rhamnolipid production was increased up to 2.6 g/L, most likely due to the inductive effect of long-chain fatty acids present in OMW (mainly oleic, palmitic, linoleic and stearic acids) on rhamnolipid production. Rhamnolipids purification was also done and the biosurfactant produced in the low-cost medium (CSL + OMW) exhibited better surface-active properties when compared with those produced in the synthetic medium, reducing the surface tension of water to 26.8 ± 0.1 mN/m, with a CMC of 280 mg/L (28.8 ± 0.2 mN/m and 460 mg/L, respectively, in the synthetic medium). These results demonstrate that rhamnolipid production is more efficient in the low-cost medium. Furthermore, to the best of the authors knowledge, this is the first experimental research that describes the utilization of CSL and CSL + OMW as substrates for the production of rhamnolipids by B. thailandensis, with very optimistic results in terms of cost and production levels.This work was sponsored by PARTEX Oil and Gas and it was supported by the Portuguese Foundation for Science and Technology(FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Cost-effective rhamnolipid production by Burkholderia thailandensis E264 using agro-industrial residues

The agro-industrial by-products corn steep liquor (CSL) and olive mill wastewater (OMW) were evaluated as low-cost substrates for rhamnolipid production by Burkholderia thailandensis E264. In a culture medium containing CSL (7.5% (v/v)) as sole substrate, B. thailandensis E264 produced 175 mg rhamnolipid/L, which is about 1.3 times the amount produced in the standard medium, which contains glycerol, peptone, and meat extract. When the CSL medium was supplemented with OMW (10% (v/v)), rhamnolipid production further increased up to 253 mg/L in flasks and 269 mg/L in a bioreactor. Rhamnolipids produced in CSL+OMW medium reduced the surface tension up to 27.1 mN/m, with a critical micelle concentration of 51 mg/L, better than the values obtained with the standard medium (28.9 mN/m and 58 mg/L, respectively). However, rhamnolipids produced in CSL+OMW medium displayed a weak emulsifying activity when compared to those produced in the other media. Whereas di-rhamnolipid congeners represented between 90 and 95% of rhamnolipids produced by B. thailandensis E264 in CSL and the standard medium, the relative abundance of mono-rhamnolipids increased up to 55% in the culture medium containing OMW. The difference in the rhamnolipid congeners produced in each medium explains their different surface-active properties. To the best of our knowledge, this is the first report of rhamnolipid production by B. thailandensis using a culture medium containing agro-industrial by-products as sole ingredients. Furthermore, rhamnolipids produced in the different media recovered around 60% of crude oil from contaminated sand, demonstrating its potential application in the petroleum industry and bioremediation.This study was funded by PARTEX Oil and Gas, the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020, and the National Science Centre (Poland), under the scope of the project 2020/37/B/NZ9/01519.info:eu-repo/semantics/publishedVersio

Biomolecular interactions of lysosomotropic surfactants with cytochrome c and its effect on the protein conformation: A biophysical approach

The molecular interactions between two single-chain lysosomotropic surfactants DMM-11 (2-Dodecanoyloxyethyl)trimethylammonium bromide) and DMPM-11 (2-Dodecanoyloxypropyl)trimethylammonium bromide) with a small heme-protein (cytochrome c (cyt-c)) in Hepes buffer (pH = 7.4) were extensively investigated by surface tension, dynamic light scattering (DLS), circular dichroism (CD) and fluorescence spectroscopy in combination with molecular dynamic simulation techniques. The results demonstrated that surfactants can destroy the hydrophobic cavity of cyt-c, make the α-helical become loose and convert it into the β-sheet structure. The interactions between surfactants and cyt-c are mainly hydrophobic. Molecular modelling approaches were also used to gather a deeper insight on the binding of lysosomotropic surfactants with cyt-c and the in silico results were found to be in good agreement with the experimental ones. This study provides a molecular basis for the applications of protein-surfactant complexes in biological, food, pharmaceutical, industrial and cosmetic systemsPolish-Portugal Executive Program for years 2017–2018 sponsored by the Polish Ministry of Science and Higher Education and by the Portuguese Foundation for Science and Technology (FCT). Additionally, FCT supported the study under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). T. Janek was supported by the National Science Centre, Poland, projects 2017/26/E/NZ9/00975 and 2018/02/X/NZ6/02201. L. R. Rodrigues acknowledges FCT for the grant SFRH/BSAB/142873/2018. E. J. Gudiña was supported by the grant UMINHO/BPD/39/2015 funded by FCT under the scope of the project UID/BIO/04469/2013info:eu-repo/semantics/publishedVersio

Effect of high pressure on surfactin production by Bacillus subtilis: implications for its application by the oil industry

Surfactin, a lipopeptide biosurfactant produced by Bacillus subtilis strains, exhibits extraordinary surface active properties, as well as stability at a wide range of temperatures and salinities, making it useful to replace the chemical surfactants in many industrial applications. The oil industry can take advantage of its application to increase the productivity of oil reservoirs, through a technology known as microbial enhanced oil recovery (MEOR). However, in order to make this technology advantageous from an economic point of view, the surfactin-producing strains must be able of growing and producing the biosurfactant inside the oil reservoirs. In this work, B. subtilis #573, isolated from an oil reservoir, was evaluated regarding its ability of producing surfactin under oxygen limited conditions at high pressure. A central composite design (CCD) was used to model the effect of pressure (3.8-46.2 bar) and temperature (35.3-46.7°C) on surfactin production. The results obtained demonstrated that pressure (in the range studied) did not exhibit a negative effect on surfactin production by this isolate, whereas temperatures higher than 45°C reduced its production. For most of the different combinations of pressure and temperature assayed, surfactin production was observed after 24 h, and the surface tension was reduced to values bellow 26.5 mN/m. At 41°C and 47 bar, B. subtilis #573 produced 31 ± 2 mg of surfactin per liter after 24 h, reducing the surface tension to 25.6 ±0.6 mN/m. These results were similar to those achieved at the same temperature at atmospheric pressure (26.0 ± 0.3 mN/m and 27 ± 3 mg surfactin/L). The surfactin produced in both cases exhibited a critical micelle concentration value around 15 mg/L, and the chemical characterization (through UHPLC-MS) demonstrated the production of similar percentages of the different surfactin isoforms (C12-, C13-, C14-, C15- and C16-surfactin) in both conditions. Finally, the applicability of B. subtilis #573 in MEOR was studied in sand-pack columns. In assays performed at 41°C and 47 bar, additional oil recoveries around 14% were obtained after 14 days in in situ assays. These results demonstrate the applicability of B. subtilis #573 in in situ oil recovery processes.info:eu-repo/semantics/publishedVersio

Sustainable exopolysaccharide production by rhizobium viscosum CECT908 using corn steep liquor and sugarcane molasses as sole substrates

Microbial exopolysaccharides (EPS) are promising alternatives to synthetic polymers in a variety of applications. Their high production costs, however, limit their use despite their outstanding properties. The use of low-cost substrates such as agro-industrial wastes in their production, can help to boost their market competitiveness. In this work, an alternative low-cost culture medium (CSLM) was developed for EPS production by Rhizobium viscosum CECT908, containing sugarcane molasses (60 g/L) and corn steep liquor (10 mL/L) as sole ingredients. This medium allowed the production of 6.1 ± 0.2 g EPS/L, twice the amount produced in the standard medium (Syn), whose main ingredients were glucose and yeast extract. This is the first report of EPS production by R.viscosum using agro-industrial residues as sole substrates. EPSCSLM and EPSSyn exhibited a similar carbohydrate composition, mainly 4-linked galactose, glucose and mannuronic acid. Although both EPS showed a good fit to the HerschelBulkley model, EPSCSLM displayed a higher yield stress and flow consistency index when compared with EPSSyn, due to its higher apparent viscosity. EPSCSLM demonstrated its potential use in Microbial Enhanced Oil Recovery by enabling the recovery of nearly 50% of the trapped oil in sand-pack column experiments using a heavy crude oil.info:eu-repo/semantics/publishedVersio

Efficient conversion of black cumin cake from industrial waste into lipopeptide biosurfactant by Pseudomonas fluorescens

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bej.2023.108981.Most biosurfactants are obtained using costly culture media, which limits their wider industrial use. In the present study, a low-cost culture medium, containing the agro-industrial residue black cumin cake, was developed for amphisin production by Pseudomonas fluorescens DSS73. By using black cumin cake as the substrate, not only was the production cost reduced but also a higher production yield was achieved. A Box-Behnken experimental design was applied to maximize lipopeptide biosurfactant production. The optimal conditions for amphisin production, such as black cumin cake (6.6%) and NaCl (8.0 mM) concentration, and cultivation time (6.5 days), were determined. Yield of amphisin production, performed in optimal conditions, reached 16.51 ± 0.49 g/L. Such high production has not been evidenced previously for Pseudomonas lipopeptide biosurfactants. Moreover, active utilization of the substrate, observed with the aid of scanning electron microscopy (SEM), documented by numerous holes and pitting on the black cumin cake surface, was confirmed. Finally, antifungal activity of amphisin against Aspergillus carbonarius was demonstrated. Hence, amphisin production by P. fluorescens was achieved with statistical optimization using an inexpensive agro-industrial by-product for the first time.This work was supported by the National Science Centre, Poland, project 2020/37/B/NZ9/01519. EJG would like to acknowledge the support of the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Biological saline produced water treatment, a sustainable process towards lipids production

Oil and gas industry is responsible for the generation of large volumes of oil-contaminated wastewaters, such as saline produced water (PW), that without proper treatment can cause environmental contamination. Alcanivorax borkumensis SK2 is a biosurfactant producer capable of degrading and converting hydrocarbons into bacterial lipids under nutrient limiting conditions (e.g. nitrogen and/or oxygen). Recently, the industrial interest in biosurfactants and bacterial lipids for biotechnological applications (e.g. bioremediation or biofuels production) is increasing. Pursuing the interest of treating and simultaneously valorizing PW, a sequencing batch airlift reactor (SBAR) strategy consisting of sequential cycles of feast and famine stages was developed. A. borkumensis SK2 was used as bioreactor inoculum and the effect of cycle duration, total petroleum hydrocarbon to nitrogen ratio (TPH/N) and dissolved oxygen (DO) concentration (7-8 mg L-1 and 2-3 mg L-1 (famine and feast stage); and 7-8/1-2 mg L-1 (famine/feast stage)) were investigated. The system provided an efficient PW treatment, achieving TPH removal efficiencies in a narrow range from 90 ± 2.1 to 96 ± 1.8 %. Intracellular lipid production increased from 0.48 to 0.74 g g-1 of cellular dry weight (CDW) with the application of higher feast stage duration and lower TPH/N ratios suggesting that nitrogen availability is the most relevant factor to promote accumulation. Under 2-3 mg L-1 and 7-8/1-2 mg L-1 of oxygen, lipid accumulation dropped to 0.50 g g-1 of CDW. Intracellular lipid profile changed according to the DO concentration. Triacylglycerols (TAG) and wax esters (WE) were accumulated under maximum and limiting DO concentrations, while polyhydroxyalkanoates (PHA) accumulation was triggered by the application of alternated DO conditions, indicating that the type of intracellular compound can be selected by manipulating the oxygen concentration in the SBAR. Extracellular lipid production (TAG and WE) was not significantly affected by none of the operational conditions applied (0.06 g L-1). Moreover, the production of a cell-bound and an extracellular glycolipid biosurfactant capable of reducing the medium surface tension from 65 to approximately 41 mN m-1 was observed. The proposed strategy showed that biological PW treatment in a SBAR can be a sustainable process through the production of added-value compounds contributing to a circular economy model.This research was supported by the Portuguese Foundation for Science and Technology (FCT I.P.) and European Regional Development Fund (ERDF) under the scope of project SaltOil+ (POCI-01- 0145- FEDER-030180) (Portugal 2020, COMPETE 2020); Rita M. Silva PhD grant (SFRH/BD/116154/2016) was funded by FCT.info:eu-repo/semantics/publishedVersio