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. KEY POINTS: • B. thailandensis produced RL using agro-industrial by-products as sole substrates • Purified RL displayed excellent surface activity (minimum surface tension 27mN/m) • Crude RL (cell-free supernatant) recovered 60% of crude oil from contaminated sand.

Characterization of levan produced by a Paenibacillus sp. isolated from Brazilian crude oil.

A levan-type fructooligosaccharide was produced by a Paenibacillus strain isolated from Brazilian crude oil, the purity of which was 98.5% after precipitation with ethanol and dialysis. Characterization by FTIR, NMR spectroscopy, GC-FID and ESI-MS revealed that it is a mixture of linear ß(2 â†’ 6) fructosyl polymers with average degree of polymerization (DP) of 18 and branching ratio of 20. Morphological structure and physicochemical properties were investigated to assess levan microstructure, degradation temperature and thermomechanical features. Thermal Gravimetric Analysis highlighted degradation temperature of 218 °C, Differential Scanning Calorimetry (DSC) glass transition at 81.47 °C, and Dynamic Mechanical Analysis three frequency-dependent transition peaks. These peaks, corresponding to a first thermomechanical transition event at 86.60 °C related to the DSC endothermic event, a second at 170.9 °C and a third at 185.2 °C, were attributed to different glass transition temperatures of oligo and polyfructans with different DP. Levan showed high morphological versatility and technological potential for the food, nutraceutical, and pharmaceutical industries.

Microbial Surfactants: Alternative to Vegetable Oil Surfactants.

Biosurfactants, surface active molecules synthesized by microorganisms, represent a promising alternative to the synthetic surfactants in many different applications. Among them, rhamnolipids have attracted considerable attention in the last years due to their extraordinary surface-active properties and biological activities. Rhamnolipids are usually synthesized by the gram-negative bacterium Pseudomonas aeruginosa as complex mixtures of different congeners. In this chapter, we describe the most common techniques that can be used for the production, recovery and purification of rhamnolipids, using two sequential chromatographic techniques to recover and separate the monorhamnolipid and dirhamnolipid congeners.

Improvement of biosurfactant production by Wickerhamomyces anomalus CCMA 0358 and its potential application in bioremediation.

In this work, biosurfactant production by Wickerhamomyces anomalus CCMA 0358 was increased through the development of an optimized culture medium using response surface methodology. The optimized culture medium contained yeast extract (4.64 g/L), ammonium sulfate (4.22 g/L), glucose (1.39 g/L) and olive oil (10 g/L). Biosurfactant production using this medium was validated both in flasks and bioreactor, and the surface tension was reduced from 49.0 mN/m up to 31.4 mN/m and 29.3 mN/m, respectively. In both cases, the highest biosurfactant production was achieved after 24 h of growth. W. anomalus CCMA 0358 demonstrated to be a fast biosurfactant producer (24 h) as compared to other yeast strains previously reported (144-240 h). The produced biosurfactant remained stable at high temperature (121 °C), NaCl concentrations as high as 300 g/L, and pH values between 6 and 12. The crude biosurfactant allowed the recovery of 20% of crude oil from contaminated sand, being a promising candidate for application in bioremediation or in the petroleum industry.

Novel bioemulsifier produced by a Paenibacillus strain isolated from crude oil.

BACKGROUND: Surface active compounds produced by microorganisms are attracting a pronounced interest due to their potential advantages over their synthetic counterparts, and to the fact that they could replace some of the synthetics in many environmental and industrial applications. RESULTS: Bioemulsifier production by a Paenibacillus sp. strain isolated from crude oil was studied. The bioemulsifier was produced using sucrose with and without adding hydrocarbons (paraffin or crude oil) under aerobic and anaerobic conditions at 40°C. It formed stable emulsions with several hydrocarbons and its emulsifying ability was not affected by exposure to high salinities (up to 300 g/l), high temperatures (100°C-121°C) or a wide range of pH values (2-13). In addition, it presented low toxicity and high biodegradability when compared with chemical surfactants. A preliminary chemical characterization by Fourier Transform Infrared Spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance (1H NMR and 13C CP-MAS NMR) and size exclusion chromatography indicated that the bioemulsifier is a low molecular weight oligosaccharide-lipid complex. CONCLUSION: The production of a low molecular weight bioemulsifier by a novel Paenibacillus strain isolated from crude oil was reported. To the best of our knowledge, bioemulsifier production by Paenibacillus strains has not been previously reported. The features of this novel bioemulsifier make it an interesting biotechnological product for many environmental and industrial applications. Graphical Abstract Novel bioemulsifier from Paenibacillus sp.

Biosurfactant production by Bacillus subtilis using corn steep liquor as culture medium.

In this work, biosurfactant production by Bacillus subtilis #573 was evaluated using corn steep liquor (CSL) as culture medium. The best results were obtained in a culture medium consisting of 10% (v/v) of CSL, with a biosurfactant production of about 1.3 g/l. To the best of our knowledge, this is the first report describing biosurfactant production by B. subtilis using CSL as culture medium. Subsequently, the effect of different metals (iron, manganese, and magnesium) on biosurfactant production was evaluated using the medium CSL 10%. It was found that for all the metals tested, the biosurfactant production was increased (up to 4.1, 4.4, and 3.5 g/l for iron, manganese, and magnesium, respectively). When the culture medium was supplemented with the optimum concentration of the three metals simultaneously, the biosurfactant production was increased up to 4.8 g/l. Furthermore, the biosurfactant exhibited a good performance in oil recovery assays when compared with chemical surfactants, which suggests its possible application in microbial enhanced oil recovery or bioremediation.

Bioconversion of agro-industrial by-products in rhamnolipids toward applications in enhanced oil recovery and bioremediation.

In this work, biosurfactant production by a Pseudomonas aeruginosa strain was optimized using low-cost substrates. The highest biosurfactant production (3.2 g/l) was obtained using a culture medium containing corn steep liquor (10% (v/v)) and molasses (10% (w/v)). The biosurfactant reduced the surface tension of water up to 30 mN/m, and exhibited a high emulsifying activity (E24=60%), with a critical micelle concentration as low as 50 mg/l. The biosurfactant produced in this alternative medium was characterized as a mixture of eight different rhamnolipid congeners, being the most abundant the mono-rhamnolipid Rha-C10-C10. However, using LB medium, nine different rhamnolipid congeners were identified, being the most abundant the di-rhamnolipid Rha-Rha-C10-C10. The rhamnolipid mixture produced in the alternative medium exhibited a better performance in removing oil from contaminated sand when compared with two chemical surfactants, suggesting its potential use as an alternative to traditional chemical surfactants in enhanced oil recovery or bioremediation.

Biosurfactant-producing and oil-degrading Bacillus subtilis strains enhance oil recovery in laboratory sand-pack columns.

Microbial Enhanced Oil Recovery (MEOR) technology uses microorganisms and their metabolites to retrieve unrecoverable oil from mature reservoirs. In situ stimulation of biosurfactant-producing and oil-degrading microorganisms reduces the capillary forces retaining the oil inside the reservoir and decreases its viscosity, thus promoting oil flow and consequently production. In this work, a sand-pack column model was designed to simulate oil recovery operations and evaluate mobilization of residual oil by the selected microorganisms. Four different hydrocarbon mixtures and three Bacillus subtilis strains isolated from crude oil samples were used. Additional oil recoveries ranged from 6 to 24% depending on the hydrocarbon mixture and microorganism used. Biosurfactant production was observed with all the microorganisms and hydrocarbon mixtures studied. The oils recovered after incubation with B. subtilis isolates showed a reduction in the percentage of long-chain n-alkanes and lower viscosity when compared with the original oils. The results obtained suggest that stimulation of the selected B. subtilis strains in situ can contribute to mobilize entrapped oil in mature reservoirs.

Characterization by electrospray ionization and tandem mass spectrometry of rhamnolipids produced by two Pseudomonas aeruginosa strains isolated from Brazilian crude oil.

In this work, biosurfactants produced by two Pseudomonas aeruginosa strains isolated from Brazilian crude oils were identified by proton nuclear magnetic resonance ((1)H NMR) and further characterized by mass spectrometry (MS) coupled with electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) analysis in positive mode and their surface activities evaluated. Mono-rhamnolipids and di-rhamnolipids were identified for both isolates, but the most abundant were found to be mono-rhamnolipids. The similarity of rhamnolipids produced by the two strains was in good agreement with their surface activities. Both biosurfactants exhibited similar aqueous solution surface tensions, high emulsification indexes and critical micelle concentration values. The results obtained show that ESI-MS and MS/MS analysis alone provide a fast and highly specific characterization of biosurfactants produced by microbial strains.

Performance of a biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants.

In this work, the biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples was characterized and its properties compared with commercially available chemical surfactants. The purified biosurfactant production yield (Y(P/X)) was 0.20 g/gcell dry weight. The surface tension (29.0 mN/m) and critical micelle concentration (40 mg/l) were found to be similar to the values previously reported for surfactin. Temperature and pH stability of the biosurfactant were also evaluated. The biosurfactant was exposed to different temperatures (20, 37 and 46°C) during two weeks and was found to be as stable as the commercial chemical surfactants Glucopone(®)215, Glucopone(®)650, Findet(®)1214N/23 and linear alkylbenzene sulfonates (LAS). Moreover, the biosurfactant subjected to 121°C for 20 min did not exhibit a significant loss of surface activity. Contrary to the commercial chemical surfactants that were found to be stable over a wide range of pH (3.0-10.0), the biosurfactant was unstable precipitating at pH values below 5.0. The emulsification indexes showed that the biosurfactant possesses equal or superior capacity to form emulsions with n-hexadecane as compared to the commercial chemical surfactants. Moreover, the anti-adhesive activity of the biosurfactant and commercial chemical surfactants was evaluated. The biosurfactant showed some activity against Staphylococcus aureus and Escherichia coli. Nevertheless, no particular trend or special effect could be assigned to the use of commercial chemical surfactants as anti-adhesives. Results gathered in this work suggest that the biosurfactant recovered from B. subtilis EG1 constitutes an interesting alternative to the commercial chemical surfactants with potential use in several industries.