Production and Recovery of Rhamnolipids Using Sugar Cane Molasses as Carbon Source

Biosurfactants were synthesized by Pseudomonas aeruginosa （P.A.）, using sugar cane molasses as carbon source. Assays were conducted in a shaker with agitation speed of 200 rpm, temperature of 38 ℃ and aeration ratio （Vm/Vf） of 0.4 and 0.6. A concentration of 3.0% was used for the carbon and energy source （molasses） and of 0.3% for the nitrogen source （NaNO3）. Samples were removed at regular times until 96 hours of cultivation. The reduction in surface tension was measured using the ring method; cell concentration was obtained by the dry mass and substrate consumption by the DNS method. The metabolite produced was extracted and quantified by the thioglycolic method. The results showed a maximum surface tension reduction of 46.57% after 60 h, 3.63 g/L of biomass after 8 h （μXmax =0.15 h^-1）, 79.60% of substrate consumption （μs= 0.67 h-1） and 4.47 g/L of rhamnolipid （μp=0.029 h^-1）.

Reuse of waste frying oil for production of rhamnolipids using Pseudomonas aeruginosa zju.u1M

In this work,rhamnolipid production was investigated using waste frying oil as the sole carbon source. By culture in shaking flasks,a naturally isolated strain synthesized rhamnolipid at concentration of 12.47 g/L and its mutant after treatment by UV light increased this productivity to 24.61 g/L. Fermentation was also conducted in a 50 L bioreactor and the productivity reached over 20 g/L. Hence,with a stable and high productive mutant strain,it could be feasible to reuse waste frying oil for rhamnolipid production on industrial scale.

Biodegradation of crude oil by Pseudomonas aeruginosa in the presence of rhamnolipids

The potential biodegradation of crude oil was assessed based on the development of a fermentative process with a strain of Pseudomonas aeruginosa which produced 15.4 g/L rhamnolipids when cultured in a basal mineral medium using glycerol as a sole carbon source. However, neither cell growth nor rhamnolipid production was observed in the comparative culture system using crude oil as the sole carbon source instead. As rhamnolipid, an effective biosurfactant, has been reported to stimulate the biodegradation of hydrocarbons, 1 g/L glycerol or 0.22 g/L rhamnolipid was initially added into the medium to facilitate the biodegradation of crude oil. In both situations, more than 58% of crude oil was degraded and further converted into accumulated cell biomass and rhamnolipids. These results suggest that Pseudomonas aeruginosa could degrade most of crude oil with direct or indirect addition of rhamnolipid. And this conclusion was further supported by another adsorption experiment, where the ad-sorption capacity of crude oil by killed cell biomass was negligible in comparison with the biologic activities of live cell biomass.

Rhamnolipids Induced by Glycerol Enhance Dibenzothiophene Biodegradation in Burkholderia sp. C3

In highly urbanized areas,pollution from anthropogenic activities has compromised the integrity of the land,decreasing soil availability for agricultural practices.Dibenzothiophene(DBT)is a heterocyclic aromatic hydrocarbon frequently found in urbanized areas,and is often used as a model chemical to study the microbial transformation of pollutants.The potential for human exposure and its health risk makes DBT a chemical of concern;thus,it needs to be environmentally managed.We utilized glycerol to stimulate Burkholderia sp.C3 in order to degrade DBT in respect to①DBT biodegradation kinetics,②bacterial growth,③rhamnolipid(RL)biosynthesis,and④RL secretion.Under an optimum glycerol-to-DBT molar ratio,the DBT biodegradation rate constants increased up to 18-fold and enhanced DBT biodegradation by 25%–30%at day 1 relative to cultivation with DBT alone.This enhancement was correlated with an increase in bacterial growth and RL biosynthesis.Proteomics studies revealed the enzymes involved in the upper and main steps of RL biosynthesis.The RL congeners Rha-C10-C10,Rha-Rha-C10-C10,Rha-Rha-C10-C12,and Rha-Rha-C12-C12 were identified in the medium supplemented with glycerol and DBT,whereas only Rha-C12-C12 was identified in cultures without glycerol or with RL inhibitors.The studies indicated that glycerol enhances DBT biodegradation via increased RL synthesis and bacterial growth.The results warrant further studies of environmental biostimulation with glycerol to advance bioremediation technologies and increase soil availability for agricultural purposes.

Milk phospholipids-based nanostructures functionalized with rhamnolipids and bacteriocin:Intrinsic and synergistic antimicrobial activity for cheese preservation

Milk fat globule membrane (MFGM) phospholipids-based nanostructures were developed and their functionalization with rhamnolipids (RLs) was carried out to enhance the preservation of cheese against resistant foodborne pathogens i.e.,Listeria monocytogenes and Escherichia coli .Void (without nisin) and nisin-loaded RLs functionalized MFGM nanostructures (RLs-MFGM-NS) were fabricated by ultrasonication-assisted self-assembly method.Cubic morphology of void and loaded RLs-MFGM-NS and pristine MFGM nanostructures (MFGM-NS) was observed under scanning electron microscopy (SEM),which indicated uniform size ranging from 43 nm (void RLs-MFGM-NS) to 194 nm (loaded RLs-MFGM-NS).FTIR analyses confirmed the electrostatic interaction,predominantly H-bonding and linkage of carboxyl ester group of MFGM with C–H group in RLs after functionalization of NS.Furthermore,quantitative antimicrobial assay on cheese slices confirmed the broad-spectrum potential of intrinsically active nanostructures (due to RLs) having synergistic activity with nisin against L.monocytogenes and E.coli .Hence,nisin-loaded RLs-MFGM-NS can be applied as promising bioactive additives for sustained preservation of cheese.

Production of rhamnolipids-producing enzymes of Pseudomonas in E. coli and structural characterization

Rhamnolipids are a class ofbiosurfactants that have a great potential to be used in industries. Five proteins/enzymes, namely RhlA, RhlB, RhlC, RhlG and RhlI, are critical for the production of rhamnolipids in Pseudomonas aeruginosa. Four of the 5 proteins except RhlC were successfully over-expressed in E. coli and three of them （RhlA, RhlB and RhlI） were purified and obtained in milligram quantities. The purified proteins were shown to be folded in solution. Homology models were built for RhlA, RhlB and RhlI. These results lay a basis for further structural and functional characterization of these proteins in vitro to favor the construction of super strains for rhamnolipids production.

Biotreatment of oily wastewater by rhamnolipids in aerated active sludge system

Oily wastewater generated by various industries creates a major ecological problem throughout the world. The tra- ditional methods for the oily wastewater treatment are inefficient and costly. Surfactants can promote the biodegradation of pe- troleum hydrocarbons by dispersing oil into aqueous environment. In the present study, we applied rhamnolipid-containing cell-free culture broth to enhance the biodegradation of crude oil and lubricating oil in a conventional aerobically-activated sludge system. At 20 ℃, rhamnolipids （11.2 mg/L） increased the removal efficiency of crude oil from 17.7% （in the absence of rham- nolipids） to 63%. At 25 ~C, the removal efficiency of crude oil was over 80% with the presence of rhamnolipids compared with 22.3% in the absence of rhamnolipids. Similarly, rhamnolipid treatment （22.5 mg/L） for 24 h at 20℃ significantly increased the removal rate of lubricating oil to 92% compared with 24% in the absence of rhamnolipids. The enhanced removal of hydrocarbons was mainly attributed to the improved solubility and the reduced interfacial tension by rhamnolipids. We conclude that a direct application of the crude rhamnolipid solution from cell culture is effective and economic in removing oily contaminants from wastewater.

Physicochemical Properties of Rhamnolipid Biosurfactant from <i>Pseudomonas aeruginosa</i>PA1 to Applications in Microemulsions

Interfacial properties rhamnolipids from an extract produced by a strain of Pseudomonas aeruginosa were analyzed in this study. The extract of rhamnolipid was characterized by surface tension in different conditions;interfacial tension with different hydrocarbons;critical micelle concentration under different pH and temperatures;particle size and emulsification capacity using laser light profiling. It was observed that the rhamnolipids extract are sensitive to variations in pH, thermostable and function as good emulsificant for emulsification of methyl methacrylate. The emulsion stability order in function of the oil phase was methyl methacrylate > emulsions of castor oil > emulsion n-heptane > emulsion toluene > emulsion hexadecane > octane emulsion. The data presented show that rhamnolipid extracts may be used to formulate stable emulsions of methyl methacrylate. This process can be used to do nano/microsphere of polymethyl methacrylate.

Biosurfactant Production in Aerobic and Anaerobic Conditions by Different Species of the Genus Pseudomonas

In a time where surface active agents are capable of reducing the energy of the bonds between water molecules by interacting with them to reduce surface tension, it would be unwise not to be able to generate these in masses. Different Pseudomonas species were grown in MSP （minimal sulphate phosphate） media containing salts, glycerol and glucose. P. aeruginosa grown aerobically in the presence of glycerol as carbon source showed the highest emulsion percentage （81.48%）, most significant decrease in surface tension （20 mN/m） and rhamnose production of 2.86 mg/mL. However, in anaerobic conditions there was no emulsion, rhamnolipid production or decrease in surface tension. The rhamnolipids were molecularly characterized using ESI-MS （electrospray ionization-mass spectrometry）, P. aeruginosa CVCM 411 is able to produce mono-rhamnolipids and di-rhamnolipids, being rhamnolipid RhC10C12.1 the predominant monomer. The specific growth rate for isolates ofP. aeruginosa and P.fluorescens in MSP are 0.6732 ht and 0.2181 h^-1, respectively. In conclusion, the formation of rhamnolipids by P. aeruginosa is linked to its growth （depending on μ）, and its ability to generate about 35% of the μmax in the presence of glucose （carbon source） and glycerol （applied as pulses）.

Stimulatory effects of biosurfactant produced by Pseudomonas aeruginosa BSZ-07 on rice straw decomposing

Biosurfactant,produced by Pseudomonas aeruginosa BSZ-07,was added to the dee straw decomposing process to enhance the production of reducing sugars.Observed by Fourier Transform InfraRed(FT-IR)and Nuclear Magnetic Resonance(NMR)analysis, the purified biosurfactant was considered as a mixture of RL1 and RL2,which are two different types of rhamnolipids.Two different adding methods,adding the purified rhamnolipid and the on-site production of it were compared.The results showed that 0.5 g/L was the optimum co...

Influence factors analysis of removing heavy metals from multiple metal-contaminated soils with different extractants

Some key factors on the heavy metals removal efficiencies were studied when soil washing technology was used in the remediation of soils contaminated by multiple heavy metals. The results show that the dissolubilities of Cu and Zn are promoted by humic acids, but Pb and Cd are inhibited by humic acids; heavy metals in the clay are more difficult to be extracted than silt; the strong acidic soils can cause the protonation of EDTA and weaken its extracting ability; EDTA is effective for extracting Pb and Cd, while oxalate (OX) is effective for extracting Cu and Zn; and biosurfactant can be used as additive to improve the removal of some particular heavy metals.

Optimizing rhamnolipid production by Pseudomonas aeruginosa ATCC 9027 grown on waste frying oil using response surface method and batch-fed fermentation

Rhamnolipid production by Pseudomonas aeruginosa ATCC 9027 with waste frying oil as sole carbon source was studied using response surface method. Cultures were incubated in shaking flask with temperature, NO3- and Mg2+ concentrations as the variables. Meanwhile, fed-batch fermentation experiments were conducted. The results show that the three variables are closely related to rhamnolipid production. The optimal cultivation conditions are of 6.4 g/L NaNO3 , 3.1 g/L MgSO4 at 32 ℃, with the maximum rhamnolipid production of 6.6 g/L. The results of fed-batch fermentation experiments show that feeding the oil in two batches can enhance rhamnolipid production. The best time interval is 72 h with the maximum rhamnolipid production of 8.5 g/L. The data are potentially useful for mass production of rhamnolipid on oil waste with this bacterium.

Enzymatic reaction of ethanol and oleic acid by lipase and lignin peroxidase in rhamnolipid(RL) reversed micelles

An environment friendly bio-surfactant of rhamnolipid(RL) was used as a solvent. The enzymatic reaction of oleic acid catalyzed by lipase and lignin peroxidase(lip) was evaluated. The optimum conditions of enzymatic reaction catalyzed by lipase(lip) were water to amphiphile molar ratio of 30(20), RL of 60(60) critical micelle concentration(CMC), pH of 7.0(3.0) and temperature of 40(30) °C, respectively. The change of enzyme conformation indicates that, for catalytic of lipase, water content is the most important factor of the enzymatic reaction of oleic acid, and p H for lip. With individual optimum conditions, the enzymatic efficiency of oleic acid catalyzed by lipase is higher than that by lip. In the presence of ethanol, the enzymatic reaction of oleic acid catalyzed by lipase suits Ping-Pong Bi-Bi mechanism. As an alternative to chemical reversed micelles, the RL reversed micelles are promising methods to enzymatic reaction of oleic acid.

Simultaneous Desorption of Polycyclic Aromatic Hydrocarbons and Heavy Metals from Contaminated Soils by Rhamnolipid Biosurfactants

Hydrophobic organic compounds(HOCs)and heavy metals(HMs)are toxins that usually coexist in natural environments.Due to the differences in their properties,it remains challenging to simultaneously remove HMs and HOCs.In this study,the removal of phenanthrene(Phe)and lead(Pb)from co-contaminated soils by single rhamnolipid(RL)and mixed RL-sophorolipid(SL)biosurfactants were evaluated via soil column experiments.Biosurfactant micelle sizes were determined by dynamic light scattering,and the mechanisms of micelle solubilization were studied.The effects of biosurfactant concentrations,pH,washing agent salinity and the ageing time of polluted soils on Phe and Pb desorption efficiencies were also assessed.The substantial removal of Phe and Pb using mixed RL-SL systems,when molar fractions of RLs were 0.7,was attributed to large mixed micelle formation and lower sorption losses of these systems.The optimal pH value was 6.0,while Phe desorption was favoured at high RLs and low ionic strengths.However,the RLs concentration and ionic strength had no obvious influence on Pb removal.In addition,both Phe and Pb desorption decreased with increased ageing of the polluted soils.Combined RL-SL biosurfactants can be effective for simultaneously removing HOCs and HMs from polluted soils.

The Environmental Impact of Polycyclic Aromatic Hydrocarbons: Mechanism of Extraction by Bio-Surfactant in a Microwave

In N’Djaména, the use and marketing of certain hydrocarbons does not comply with any standard in force or with regulations provided for this purpose. Their evaporation and unregulated release into the wild significantly affects the ecosystem. The present work consists in developing a method of extraction from sediments polluted by bio surfactant (rhamnolipid) assisted by microwaves. The goal here is to look for the presence of polycyclic aromatic hydrocarbons in polluted sediments. The rhamnolipid used consists of monorhamnolipids and di-rhamnolipids, its emulsion index is 64.66% and is composed of saturated and unsaturated fatty acids having carbon numbers ranging from 12 to 18. We used the microwave micellar extraction process. It was made by keeping the concentration values fixed at 0.15 g/L and the power at 400 W and more. We observed a positive interaction of the rhamnolipid concentration factors and the power of the microwave to obtain the optimal conditions at the time of 50 S, at the concentration of 0.16 g/L and at the power of 443 W for a rate of optimal extraction of 0.91%. Chromatographic analysis by GC-MS of the optimum extracts allowed us to identify twelve (12) C10 to C43 n-alkanes and eight (8) PAHs. It emerges from this analysis that the rhamnolipid extracts seven (7) Polycyclic Aromatic Hydrocarbons in equivalent proportions while the tween 80 extracts only four (4) Polycyclic Aromatic Hydrocarbons with a high proportion (80.02%) of benzo [a] antracene.

Combination of rhamnolipid and biochar in assisting phytoremediation of petroleum hydrocarbon contaminated soil using Spartina anglica

Biochar (BC) and rhamnolipid (RL) is used in bioremediation of petroleum hydrocarbons,however,the combined effect of BC and RL in phytoremediation has not been studied until now.In this paper,the phytoremediation of petroleum hydrocarbon-contaminated soil using novel plant Spartina anglica was enhanced by the combination of biochar (BC) and rhamnolipid (RL).Samples of petroleum-contaminated soil (10,30 and 50 g/kg) were amended by BC,BC+ RL and rhamnolipid modified biochar (RMB),respectively.After 60 day's cultivation,the removal rate of total petroleum hydrocarbons (TPHs) for unplanted soil (UP),planted soil (P),planted soil with BC addition (P-BC),planted soil with BC and RL addition (P-BC + RL) and planted soil with addition ofRMB (P-RMB) were 8.6%,19.1%,27.7%,32.4% and 35.1% in soil with TPHs concentration of 30 g/kg,respectively.Compared with UP,the plantation of Spartina anglica significantly decreased the concentration of C8-14 and tricyclic PAHs.Furthermore,the application of BC and RMB alleviated the toxicity of petroleum hydrocarbons to Spartina anglica via improving plant growth with increasing plant height,root vitality and total chlorophyll content.High-throughput sequencing result indicated that rhizosphere microbial community of Spartina anglica was regulated by the application of BC and RMB,with increase of bacteria and plant mycorrhizal symbiotic fungus in biochar and RMB amended soil.

Effects of a Biosurfactant and a Synthetic Surfactant on Phenanthrene Degradation by a Sphingomonas Strain

A novel phenanthrene (PHE)-degrading strain named as Sphingomonas sp.GF2B was isolated and identified from a farmland soil.Effects of a synthetic surfactant (Tween-80) and a rhamnolipid biosurfactant on PHE degradation by Sphin- gomonas sp.GF2B were investigated at different concentrations of the surfactants.The results showed that Sphingomonas sp.GF2B was able to mineralize up to 83.6% of PHE within 10 days without addition of surfactants.The addition of Tween-80 to the reaction medium inhibited greatly PHE biodegradation,with only 33.5% of PHE degraded.However,the biosurfactant facilitated PHE biodegradation,with up to 99.5% of PHE degraded.The preferential utilization of PHE as a carbon source and the enhanced solubility of PHE by the biosurfactant were likely responsible for the higher biodegra- dation efficiency of PHE in the presence of the biosurfactant.Therefore,it could be concluded that the application of the biosurfactant to PHE-contaminated soils was perhaps a feasible way to facilitate the PHE biodegradation.

Rhamnolipid synthesis and production with diverse resources

Rhamnolipids are one of the most effective biosurfactants that are of great interest in industrial applications such as enhancing oil recovery, health care, cosmetics, pharmaceutical processes, food processing, detergents for protein folding, and bioremediation due to their unique characteristics such as low toxicity, surface active property to reduce surface/interfacial tensions, and excellent biodegradability. The genes and metabolic path- ways for rhamnolipid synthesis have been well elucidated, but its cost-effective production is still challenging. Pseudomonas aeruginosa, the most powerful rhamnolipid producer, is an opportunistic pathogen, which limits its large scale production and applications. Rhamnolipid production using engineered strains other than Pseudomo- nas aeruginosa such as E. coli and Pseudomonas putida has received much attention. The highest yield of rhamnolipids is achieved when oil-type carbon sources are used, but using cheaper and renewable carbon sources such as lignocellulose would be an attractive strategy to reduce the production cost of rhamnolipids for various industrial applications.

Enhanced biodegradation of chlorobenzene via combined Fe^(3+) and Zn^(2+) based on rhamnolipid solubilisation

Biotrickling filters(BTFs) for hydrophobic chlorobenzene(CB) purification are limited by mass transfer and biodegradation. The CB mass transfer rate could be improved by 150 mg/L rhamnolipids. This study evaluated the combined use of Fe^(3+) and Zn^(2+) to enhance biodegradation in a BTF over 35 day. The effects of these trace elements were analysed under different inlet concentrations(250, 600, 900, and 1200 mg/L) and empty bed residence times(EBRTs;60, 45, and 32 sec). Batch experiments showed that the promoting effects of Fe^(3+)/Zn^(2+) on microbial growth and metabolism were highest for 3 mg/L Fe^(3+) and 2 mg/L Zn^(2+), followed by 2 mg/L Zn^(2+), and lowest at 3 mg/L Fe^(3+). Compared to BTF in the absence of Fe^(3+) and Zn^(2+), the average CB elimination capacity and removal efficiency in the presence of Fe^(3+) and Zn^(2+) increased from 61.54 to 65.79 g/(m 3 hr) and from 80.93% to 89.37%, respectively, at an EBRT of 60 sec. The average removal efficiency at EBRTs of 60, 45, and 32 sec increased by 2.89%, 5.63%, and 11.61%, respectively. The chemical composition(proteins(PN), polysaccharides(PS)) and functional groups of the biofilm were analysed at 60, 81, and 95 day. Fe^(3+) and Zn^(2+) significantly enhanced PN and PS secretion, which may have promoted CB adsorption and biodegradation. High-throughput sequencing revealed the promoting effect of Fe^(3+) and Zn^(2+) on bacterial populations. The combination of Fe^(3+) and Zn^(2+) with rhamnolipids was an efficient method for improving CB biodegradation in BTFs.

Leaching of rare earth elements from contaminated soils using saponin and rhamnolipid bio-surfactant

The effective leaching of rare earth elements（La, Ce, Y and Eu） from simulated contaminated soil using bio-surfactant was investigated in a lab-scale column leaching experiment, where anionic biosurfactant rhamnolipid and non-ionic biosurfactant saponin were used as washing solutions. Soil properties and the rare earth element fractions were analysed to define the effect of leaching on soil and elemental speciation. Column leaching results showed that saponin solution was more effective than rhamnolipid in the removal of the four rare earth elements tested, with the accumulative removal efficiency of La, Ce, Y and Eu following flushing with 400 mL of 25 g/L saponin, reaching 35.258%, 26.072%, 31.476% and 30.849%, respectively. The change in REE speciation showed that REE removed from soils were mainly derived from the acid-soluble and residual fractions released when rhamnolipid solution was used as a leaching agent. However, for saponin leaching, removed REE amounts were derived from acid-soluble and reducible fractions. Complexation interactions were identified between saponin and REEs, according to infrared spectroscopy and ion exchange data, with saponin complexing with La, Ce, Y, and Eu at a complex ratio of 1：1.