Reduced-Cost Production of Sophorolipids by Starmerella bombicola CGMCC1576 Grown on Cottonseed Molasses and Cottonseed Oil-Based Medium.

A large-scale application of sophorolipids (SLs) was blocked by their high production cost. One feasible way to reduce the cost of SL production is to develop cheap feedstocks as the substrates for SL fermentation. In the present work, cottonseed molasses (CM), a waste from raffinose production, was used as the hydrophilic substrate;, and cottonseed oil (CO) was used as a hydrophobic substrate for SL production by Starmerella bombicola CGMCC 1576. The primary optimization of carbon sources, nitrogen source and inorganic salts, produced 57.6 ± 2.3 g/L of total SLs and 24.0 ± 1.2 g/L of lactonic SLs on CM and CO, almost equal to the titer of SLs produced from glucose and oleic. A response surface method was applied to optimize the fermentation medium for growth and SL production of S. bombicola. The production of total SLs reached 58.4 ± 3.4 g/L, and lactonic SLs were elevated to more than 25.0 ± 1.9 g/L. HPLC-MS analysis showed that the compositions of SLs produced by S. bombicola on CM and CO were very similar to those on glucose and oleic acid. These results suggested that cottonseed molasses and cottonseed oil can be used as renewable cheap substrates for the reduced-cost production of SLs.

Kinetic modeling and quasi-economic analysis of fermentative glycolipopeptide biosurfactant production in a medium co-optimized by statistical and neural network approaches.

This study presents the kinetics of production of a glycolipopeptide biosurfactant in a medium previously co-optimized by response surface and neural network methods to gain some insight into its volumetric and specific productivities for possible scale-up towards industrial production. Significant kinetic parameters including maximum specific growth rate, µmax, specific substrate consumption rate, qs and specific biosurfactant yield, Yp/x were determined from logistic model parameters after comparison with other kinetic models. Results showed that bio-catalytic rates of lipase and urease reached exponential values within the first 12 h of fermentation leading to high specific rates of substrate consumption and bacterial growth. Volumetric biosurfactant production reached significantly high levels during prolonged stationary growth and specific urease activity. This suggests that glycolipopeptide biosynthesis may proceed through stationary phase transpeptidation of the glycolipid base. A high cross-correlation coefficient of 0.950 confirmed that substrate consumption and glycolipopeptide production occurred contemporaneously during the 66-h fermentation. The maximum biosurfactant concentration of 132.52 g/L, µmax of 0.292 h-1, qp of 1.674 g/gDCW/h, rp of 2.008 g/(Lh) and Yp/x of 4.413 g/g predicted by the selected logistic model and a unit cost of €0.57/g glycolipopeptide in the optimized medium may lead to technical and economic benefits.

Potential use of biochar and rhamnolipid biosurfactant for remediation of crude oil-contaminated coastal wetland soil: Ecotoxicity assessment.

Remediation of wetland soils contaminated with petroleum hydrocarbons is a challenging task. Biosurfactant and biochar have been used in oil remediation. However, little is known about the ecotoxicity of these materials when applied in wetland ecosystems. In this study, the ecotoxicity of biochar and rhamnolipid (RL) biosurfactant as crude oil remediation strategies in a Louisiana wetland soil was investigated. A pot experiment was set up with wetland soil treated with/without crude oil followed by subjecting to application of 1% biochar and various levels of RL ranging from 0.1% to 1.4%. The ecotoxicity was evaluated regarding to high plant (S. Alterniflora), algae, and soil microbes. Specifically, after a 30-day growth in a controlled chamber, plant biomass change as well as shoot/root ratio was measured. Algae growth was estimated by quantifying chlorophyll by spectrometry following separation, and soil microbial community was characterized by phospholipid fatty acids analysis. Results showed that plant can tolerate RL level up to 0.8%, while algae growth was strongly inhibited at RL > 0.1%. Algal biomass was significantly increased by biochar, which offset the negative impact of oil and RL. Additionally, soil microbial community shift caused by crude oil and RL was alleviated by biochar with promoting Gram-positive bacteria, actinomycetes, and arbuscular mycorrhizal fungi. Overall, this study shows that integrated treatment of biochar and RL has the lowest ecotoxicity to plant and algae when used in oil remediation of contaminated wetland soils.

Enhanced volatile fatty acid production from excess sludge by combined free nitrous acid and rhamnolipid treatment.

VFA production from excess sludge (ES) was greatly enhanced by a low-cost and high-efficient treatment: 0.67mg/L free nitrous acid (FNA) pretreatment combined with 0.04g/g TSS rhamnolipid (RL) addition (FNA+RL), which significantly shortened fermentation time to 3days and increased VFA production to 352.26mgCOD/g VSS (5.42 times higher than raw ES). Propionic and acetic acids were the two leading components (71.86% of the total VFA). Mechanism investigation manifested FNA+RL improved the biodegradability of ES, achieved positive synergetic effect on solubilization, hydrolysis and acidification efficiencies, and inhibited methanation. Microbial community distribution further explained the above phenomena. The bacteria related to polysaccharides/protein utilization and VFA generation, including Clostridium, Megasphaera and Proteiniborus, were mainly observed in FNA+RL, whereas gas-forming bacteria Anaerolineae and acid-consuming bacteria Proteobacteria were assuredly suppressed. Besides, Propionibacterineae associated with propionic acid generation was exclusively enriched in sole RL and FNA+RL.

Utilization of Paneer Whey Waste for Cost-Effective Production of Rhamnolipid Biosurfactant.

The present study aimed at isolating rhamnolipid biosurfactant-producing bacteria that could utilize paneer whey, an abundant waste source as sole medium for the production purpose. Pseudomonas aeruginosa strain, SR17, was isolated from hydrocarbon-contaminated soil that could efficiently utilize paneer whey for rhamnolipid production and reduce surface tension of the medium from 52 to 26.5 mN/m. The yield of biosurfactant obtained was 2.7 g/l, upgraded to 4.8 g/l when supplemented with 2 % glucose and mineral salts. Biochemical, FTIR, and LC-MS analysis revealed that extracted biosurfactant is a combination of both mono and di-rhamnolipid congeners. The critical micelle concentration (CMC) was measured to be 110 mg/l. Emulsification activity of the biosurfactant against n-hexadecane, olive oil, kerosene, diesel oil, engine oil, and crude oil were found to be 83, 88, 81, 92, 86, and 100 %, respectively. The rhamnolipid was detected to be non-toxic against mouse fibroblastic cell line L292.

Glycolipid biosurfactants: main properties and potential applications in agriculture and food industry.

Glycolipids, consisting of a carbohydrate moiety linked to fatty acids, are microbial surface active compounds produced by various microorganisms. They are characterized by high structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Rhamnolipids, trehalolipids, mannosylerythritol lipids and cellobiose lipids are among the most popular glycolipids. They have received much practical attention as biopesticides for controlling plant diseases and protecting stored products. As a result of their antifungal activity towards phytopathogenic fungi and larvicidal and mosquitocidal potencies, glycolipid biosurfactants permit the preservation of plants and plant crops from pest invasion. Also, as a result of their emulsifying and antibacterial activities, glycolipids have great potential as food additives and food preservatives. Furthermore, the valorization of food byproducts via the production of glycolipid biosurfactant has received much attention because it permits the bioconversion of byproducts on valuable compounds and decreases the cost of production. Generally, the use of glycolipids in many fields requires their retention from fermentation media. Accordingly, different strategies have been developed to extract and purify glycolipids. © 2016 Society of Chemical Industry.

Biotechnological opportunities in biosurfactant production.

In the recent years, biosurfactants proved to be an interesting alternative to petrochemically derived surfactants. Two classes of biosurfactants, namely glycolipids and lipopeptides, have attracted significant commercial interest. Despite their environmental advantages and equal performance, commercialization of these molecules remains a challenge due to missing acquaintance of the applicants, higher price and lack of structural variation. The latter two issues can partially be tackled by screening for novel and better wild-type producers and optimizing the fermentation process. Yet, these traditional approaches cannot overcome all hurdles. In this review, an overview is given on how biotechnology offers opportunities for increased biosurfactant production and the creation of new types of molecules, in this way enhancing their commercial potential.

Integrating ReSET with glycosyl iodide glycosylation in step-economy syntheses of tumor-associated carbohydrate antigens and immunogenic glycolipids.

Carbohydrates mediate a wide range of biological processes, and understanding these events and how they might be influenced is a complex undertaking that requires access to pure glycoconjugates. The isolation of sufficient quantities of carbohydrates and glycolipids from biological samples remains a significant challenge that has redirected efforts toward chemical synthesis. However, progress toward complex glycoconjugate total synthesis has been slowed by the need for multiple protection and deprotection steps owing to the large number of similarly reactive hydroxyls in carbohydrates. Two methodologies, regioselective silyl exchange technology (ReSET) and glycosyl iodide glycosylation have now been integrated to streamline the synthesis of the globo series trisaccharides (globotriaose and isoglobotriaose) and α-lactosylceramide (α-LacCer). These glycoconjugates include tumor-associated carbohydrate antigens (TACAs) and immunostimulatory glycolipids that hold promise as immunotherapeutics. Beyond the utility of the step-economy syntheses afforded by this synthetic platform, the studies also reveal a unique electronic interplay between acetate and silyl ether protecting groups. Incorporation of acetates proximal to silyl ethers attenuates their reactivity while reducing undesirable side reactions. This phenomenon can be used to fine-tune the reactivity of silylated/acetylated sugar building blocks.

Production and characterization of rhamnolipid biosurfactant from waste frying coconut oil using a novel Pseudomonas aeruginosa D.

AIM: To improve biosurfactant production economics by the utilization of potential low-cost materials. METHODS AND RESULTS: In an attempt to utilize cost-effective carbon sources in the fermentative production of biosurfactants, various pure and waste frying oils were screened by a standard biosurfactant producing strain. Considering the regional significance, easy availability and the economical advantages, waste frying coconut oil was selected as the substrate for further studies. On isolation of more competent strains that could use waste frying coconut oil efficiently as a carbon source, six bacterial strains were isolated on cetyltrimethyl ammonium bromide-methylene blue agar plate, from a soil sample collected from the premises of a coconut oil mill. Among these, Pseudomonas aeruginosa D was selected as the potential producer of rhamnolipid. Spectrophotometric method, TLC, methylene blue active substance assay, drop collapse technique, surface tension measurement by Du Nouy ring method and emulsifying test confirmed the rhamnolipid producing ability of the selected strain and various process parameters were optimized for the production of maximum amount of biosurfactant. Rhamnolipid components purified and separated by ethyl acetate extraction, preparative silica gel column chromatography, HPLC and TLC were characterized by fast atom bombardment mass spectrometry as a mixture of dirhamnolipids and monorhamnolipids. The rhamnolipid homologues detected were Rha-Rha-C(10) -C(10) , Rha-C(12) -C(10) and Rha-C(10) -C(8) /Rha-C(8) -C(10) . CONCLUSION: These results indicated the possibility of waste frying coconut oil to be used as a very effective alternate substrate for the economic production of rhamnolipid by a newly isolated Ps. aeruginosa D. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study throws light on the alternate use of already used cooking oil as high-energy source for producing a high value product like rhamnolipid. This would provide options for the food industry other than the recycling and reuse of waste frying oils in cooking and also furthering the value of oil nuts.

Rhamnolipid surfactants: alternative substrates, new strategies.

This chapter concentrates on the various possibilities of using alternative substrates and new strategies. Such strategies include an integrated production system to reduce the environmental impact and an attempt to minimize residues, which reinforces socio-economic and region-structural development. Additionally, we offer an overview of the physicochemical and biological properties of rhamnolipid surfactants associated with the applications of these molecules in different circumstances.

Kinetics of growth and enhanced sophorolipids production by Candida bombicola using a low-cost fermentative medium.

In this study, effect of various parameters on sophorolipid (SL) production by the yeast Candida bombicola was investigated for the enhancing of its production by employing L18 orthogonal array design of experiments. At optimum conditions of sugarcane molasses 50 g l(-1), soybean oil 50 g l(-1), inoculum size 5% (v/v), temperature 30 degrees C, inoculum age 2 days, and agitation 200 rpm, the yeast produced almost equal amounts of the product in batch shake flasks and in a 3-l fermentor without any pH control (45 and 47 g l(-1), respectively). However, the yield increased to 60 g l(-1) in the fermentor under controlled pH environment. Time course of SL production, yeast biomass growth, and utilization of sugarcane molasses and soybean oil at these optimized conditions were fitted to existing kinetic models reported in the literature. Estimated kinetic parameters from these models suggested that conventional medium containing glucose can very well be replaced with the present low-cost fermentative medium.

Rapid analysis of long-chain glycolipids in heterocystous cyanobacteria using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry.

Under nitrogen-depleted conditions, N2-fixing cyanobacteria of the order Nostocales and Stigonematales differentiate vegetative cells into heterocysts. The cell envelope of these specialized cells contains unique glycolipids, consisting of a sugar moiety glycosidically bound to long-chain diols, triols and hydroxyketones. Only few reports have been published on these glycolipids in cultured cyanobacteria and none has reported them in natural environments. Here we show that heterocyst glycolipids can be rapidly and sensitively analyzed using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS2). Positive ion mass spectra of the glycolipids consisted of protonated molecules and diagnostic product ions, indicating losses of sugar groups as well as hydroxyl and carbonyl functionalities from an alkyl chain. Using this method, heterocyst glycolipids were for the first time identified in a natural ecosystem, i.e., a microbial mat from the North Sea barrier island Schiermonnikoog, The Netherlands. This technique will facilitate the quick screening of cyanobacterial cultures and natural environments for the presence of heterocyst glycolipids, which may aid in assessing the role of heterocystous cyanobacteria in the global nitrogen cycle.

Production, characterization, and properties of sophorolipids from the yeast Candida bombicola using a low-cost fermentative medium.

The yeast Candida bombicola produces biosurfactant with properties akin to those of sophorolipid (SL) group of compounds. In the present work, the yeast was shown to produce 63.7 g l(-1) SL when grown on a cheap fermentative medium containing sugarcane molasses, yeast extract, urea, and soybean oil. The partially purified SL was characterized and confirmed by Fourier-transform infrared (FT-IR) spectroscopy, 1H and 13C nuclear magnetic resonance (NMR) and liquid chromatography-mass spectroscopy (LC-MS) analysis. The critical micelle concentration (CMC) and minimum surface tension of the produced SL in aqueous solution were found to be 59.43 mg l(-1) and 34.15 m Nm(-1), respectively. The emulsification activity and stability with kerosene oil and organic solvents viz. xylene, benzene, and hexadecane were also tested using the produced SL, which yielded better results compared to those reported in the literature.

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials.

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.

Conformational studies on a unique bis-sulfated glycolipid using NMR spectroscopy and molecular dynamics simulations.

The time-averaged solution conformation of a unique bis-sulfated glycolipid (HSO3)2-2,6Manalpha-2Glcalpha-1-sn-2,3-O-alkylglycerol , was studied in terms of the torsional angles of two glycosidic linkages, phi (H1-C1-O-Cx) and psi (C1-O-Cx-Hx), derived from heteronuclear three-bond coupling constants (3JC,H), and inter-residual proton-proton distances from J-HMBC 2D and ROESY experiments, respectively. The dihedral angles of Glcalpha1Gro in glycolipids were determined for the first time. The C1-C4 diagonal line of the alpha-glucose ring makes an angle of approximately 120 degrees with the glycerol backbone, suggesting that the alpha-glucose ring is almost parallel to the membrane surface in contrast with the perpendicular orientation of the beta-isomer. Furthermore, minimum-energy states around the conformation were estimated by Monte Carlo/stochastic dynamics (MCSD) mixed-mode simulations and the energy minimization with assisted model building and energy refinement (AMBER) force field. The Glcalpha1Gro linkage has a single minimum-energy structure. On the other hand, three conformers were observed for the Manalpha2Glc linkage. The flexibility of Manalpha2Glc was further confirmed by the absence of inter-residual hydrogen bonds which were judged from the temperature coefficients of the chemical shifts, ddelta/dT (-10-3 p.p.m. degrees C-1), of hydroxy protons. The conformational flexibility may facilitate interaction of extracellular substances with both sulfate groups.

Novel polar lipid composition of Clostridium innocuum as the basis for an assessment of its taxonomic status.

The extractable polar lipids of Clostridium innocuum have been shown to consist of glycosyldiradylglycerols, phospholipids and phosphoglycolipids. The major glycosyldiradylglycerols are D-Glcp(alpha 1-3)radyl2Gro and D-Galp(alpha 1-2)D-Glcp(alpha 1-3)radyl2Gro. Both glycolipids have some 1-O-(alk-1-enyl)-2-O-acyl species, in addition to diacyl species. The phospholipids include bisphosphatidylglycerol (cardiolipin), lysocardiolipin and phosphatidylglycerol (PG). In addition, several novel lipids have been found, including a PG acetal of cardiolipin plasmalogen, smaller amounts of a lyso form of this lipid, a PG acetal of PG plasmalogen, and two phosphoglycolipids, which represent 65% of total polar lipids. The latter have been identified as 2'-amino-1',3'- dihydroxypropane-3'-P-6-D-Galp(alpha 1-2)D-Glcp(alpha 1-3)radyl2Gro and a derivative of this lipid containing an acyl chain esterified to O-6 of the glucopyranosyl ring. Based on rRNA sequence data, C. innocuum is considered to be a relative of the mycoplasmas. Its unique lipid composition permits an assessment of the taxonomic status of C. innocuum, since the lipid amphiphiles display marked differences from those of Acholeplasma laidlawii.