This ability has been reported by Al-Tahhan et al. Results of their study demonstrated that rhamnolipid, at very low concentration, caused release of lipopolysaccharide LPS from the outer membrane resulting in an increase of cell surface hydrophobicity. In contrast, Sotirova et al. However, all of the changes in the structure of the bacterial cell surface cause increase of accessibility of hydrocarbons to microbial cells.
The extensive production and use of hydrocarbons has resulted in widespread environmental contamination by these chemicals. Due to their toxicity, persistent and negative influence on living organisms it is important to clean-up the polluted sites.
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Hydrocarbons, as the hydrophobic organic chemicals, exhibit limited solubility in groundwater and tend to partition to the soil matrix. A promising method that can improve bioremediation effectiveness of hydrocarbon contaminated environments is the use of biosurfactants. They can enhance hydrocarbon bioremediation by two mechanisms. The first includes the increase of substrate bioavailability for microorganisms, while the other involves interaction with the cell surface which increases the hydrophobicity of the surface allowing hydrophobic substrates to associate more easily with bacterial cells [ 38 ].
By reducing surface and interfacial tensions, biosurfactants increase the surface areas of insoluble compounds leading to increased mobility and bioavailability of hydrocarbons. In consequence, biosurfactants enhance biodegradation and removal of hydrocarbons. Addition of biosurfactants can be expected to enhance hydrocarbon biodegradation by mobilization, solubilization or emulsification Figure 3 [ 34 , 39 — 43 ].
Mechanisms of hydrocarbon removal by biosurfactants depending on their molecular mass and concentration [ 11 , 42 ]. The mobilization mechanism occurs at concentrations below the biosurfactant CMC. In turn, above the biosurfactant CMC the solubilization process takes place. At these concentrations biosurfactant molecules associate to form micelles, which dramatically increase the solubility of oil. The hydrophobic ends of biosurfactant molecules connect together inside the micelle while the hydrophilic ends are exposed to the aqueous phase on the exterior. Consequently, the interior of a micelle creates an environment compatible for hydrophobic organic molecules.
The process of incorporation of these molecules into a micelle is known as solubilization [ 42 ]. Emulsification is a process that forms a liquid, known as an emulsion, containing very small droplets of fat or oil suspended in a fluid, usually water. The high molecular weight biosurfactants are efficient emulsifying agents.
They are often applied as an additive to stimulate bioremediation and removal of oil substances from environments. In the current literature, the latest advantages of the role of biosurfactants in interaction between hydrocarbons and microorganisms are presented. Franzetti et al. High cell-hydrophobicity allows microorganisms to directly contact oil drops and solid hydrocarbons while low cell hydrophobicity permits their adhesion to micelles or emulsified oils [ 17 ].
They discuss three mechanisms of interaction between microorganisms and hydrocarbons: access to water-solubilized hydrocarbons, direct contact of cells with large oil drops and contact with pseudosolubilized or emulsified oil. The authors suggest that during the different growth stages of microorganisms, biosurfactants can change hydrocarbon accession modes. In their studies, they observed that Gordonia sp.
The recent report by Cameotra and Singh [ 45 ] throws more light on the uptake mechanism of n -alkane by Pseudomonas aeruginosa and the role of rhamnolipids in the process. The authors reported a new and exciting research for hydrocarbon uptake involving internalization of hydrocarbon inside the cell for subsequent degradation. Biosurfactant action dispersed hexadecane into microdroplets, increasing the availability of the hydrocarbon to the bacterial cells.
The electron microscopic studies indicated that uptake of the biosurfactant-coated hydrocarbon droplets occurred. This mechanism was not earlier visually reported in bacterial modes for hydrocarbon uptake. Although much work has been done by many groups to explain the role of biosurfactants in the degradation of water immiscible substrates, most processes still remain unclear. Obayori et al. LP1 strain on crude oil and diesel. The results obtained confirmed the ability of strain LP1 to metabolize the hydrocarbon components of crude and diesel oil. They reported Biodegradative properties of biosurfactant producing Brevibacterium sp.
Biosurfactants-Types, Sources and Applications - SciAlert Responsive Version
PDM-3 strain were tested by Reddy et al. They reported that this strain could degrade Kang et al. Their results indicated that sophorolipid may have potential for facilitating the bioremediation of sites contaminated with hydrocarbons having limited water solubility and increasing the bioavailability of microbial consortia for biodegradation. The effective microbiological method in bioremediation of hydrocarbon polluted sites is the use of biosurfactant producing bacteria without necessarily characterizing the chemical structure of the surface active compounds.
The cell free culture broth containing the biosurfactants can be applied directly or by diluting it appropriately to the contaminated site. The other benefit of this approach is that the biosurfactants are very stable and effective in the culture medium that was used for their synthesis. The usefulness of biosurfactant producing strains in bioremediation of sites highly contaminated with crude petroleum-oil hydrocarbons was confirmed by Das and Mukherjee [ 50 ].
The ability of three biosurfactant producing strains: Bacillus subtilis DM, Pseudomonas aeruginosa M and Pseudomonas aeruginosa NM to remediate petroleum crude-oil contaminated soil samples was investigated by treating the soil samples with aqueous solutions of biosurfactants obtained from the respective bacteria strains. Additionally, the tested soil was inoculated with mineral-salts media containing a specified amount of Bacillus subtilis DM or Pseudomonas aeruginosa M and NM strains.
To determine the extent of biodegradation, the soil-phase total petroleum hydrocarbons TPH concentrations were analyzed after days and compared to a control where the soil was treated with un-inoculated medium. Bioagumentation of studied soil with P. Joseph and Joseph [ 51 ] separated the oil from the petroleum sludge by induced biosurfactant production by bacteria. Petroleum sludge is generated in significant amount in the refineries during crude oil processing. Crude oil is usually stored in storage tanks.
Pollutants present in the oil are deposited at bottom of the tank. During cleaning of the tank the sludge is recovered and is treated as a waste. In this study the sludge was inoculated directly with Bacillus sp. Un-inoculated sludge was also taken as a control. Upon inoculation of the supernatant to the sludge slurry, oil separation and reduction of TPH was observed. The oil separation process was slow initially in the test supplied with the fresh inoculation of the bacterium compared to the samples inoculated with the supernatant, but the residual TPH of both became equal within 48 h.
The efficiency of removal of the various isolates ranged from Therefore, it has been observed that the biosurfactant produced by the primary inoculum remained in the supernatant and it was enough to continue the reaction. The biosurfactant displayed the property to reduce surface and interfacial tensions in both aqueous and hydrocarbon mixtures and hence had potential for oil recovery. Biosurfactants have often been used to enhance bioavailability and biodegradation of hydrophobic compounds but there is little knowledge available about the effect of simultaneous emulsifier production on biodegradation of complex hydrocarbon mixtures.
Nievas et al. Bilge waste is a hazardous waste composed of a mixture of sea-water and hydrocarbon residue, where n -alkanes, resolvent total hydrocarbons and unsolvent complex mixture are the main constituents. Unsolvent complex mixture principally is composed by branched and cyclic aliphatic hydrocarbons and aromatic hydrocarbons, which usually show the greatest resistance to biodegradation. In their studies, they investigated the biodegradation of an oily bilge wastes by an emulsifier-producing microbial consortium.
Barkay et al. First, they studied capacity of strain F to grow and produce bioemulsifier in the presence of different hydrocarbon compounds. They observed that all analyzed hydrocarbons supported the growth of F strain and the production of V bioemulsifier. The ability of the analyzed strain to remove polycyclic aromatic hydrocarbons was investigated during the growth of this strain for 96 h in liquid medium supplemented with naphthalene, phenanthrene, fluoranthene and pyrene.
Biosurfactants in industry
After the experiment, the obtained residual concentrations of fluoranthene Efficiency of strain F in removing PAHs confirmed its potential applicability in oil bioremediation technology. The EPS could increase the hydrophobicity of the bacterial cell surface and also neutralize the surface charge of the cells. Soil washing technology is characterized by chemico-physical properties of the biosurfactant and not by their effect on metabolic activities or changes in cell-surface properties of bacteria [ 55 ]. However, the processes may enhance the bioavailability for bioremediation.
Aqueous solutions of biosurfactants can be also used to release compounds characterized by low solubility from soil and other media in process called washing. Urum et al. They observed that sophorolipid had a higher soil washing efficiency that any other tested nonionic surfactants except Tween This could be caused by high hydrophilic-lipophilic balance HLB of Tween It appeared that surfactants with a higher HLB resulted in better solubility of 2-methylnapthalene.
Lai et al. As a result, they observed that addition of 0. These results indicated that among four bio surfactants, rhamnolipid and surfactin showed superior performance on TPH removal, compared to synthetic surfactants. The two biosurfactants examined in this work have the potential to be used as biostimulation agents for bioremediation of oil-polluted soils.
The work represents the first study on the potential applications of surface-active compounds produced by Gordonia sp. In the previous work, surface-active compounds produced by Gordonia sp. The bacterial strain grew on aliphatic hydrocarbons and produced two different types of surface active compounds: extracellular bioemulsan and cell-bound biosurfactant. Bioremediation results showed that the bioemulsans produced by Gordonia sp. On the other hand, the authors obtained the best results in soil washing of hydrocarbons.
The study presented by Franzetti et al.
The BS29 bioemulsans were also able to remove metals Cu, Cd, Pb, Zn, Ni , but their potential in the process was lower than rhamnolipids. The aim of the research work reported by Kildisas et al. The described technology was based on bioremediation or phytoremediation principles and used physical-chemical treatment by washing the contaminated soil. The complex technology consisted of two stages: at the first stage, the migrating fraction of pollutants was separated from soil using biosurfactants; at the second stage, the remaining not migrating fraction was rendered harmless using biodegradation.
Phytoremediation was also applied to enhance soil quality. The completed clean up complex technology is presented by Kildisas et al. The presented technology consisted of washing of the migration fraction by application of biosurfactants, separation of water, oil and soil, biodegradation of residual non-migrating oil fraction by use of specific bacteria with potential to degrade the crude oil and oil products, and phytoremediation. The pilot plant for washing the contaminated soil was designed and constructed in a space of m 2 in which m 3 of contaminated soil was cleaned up.
After degradation, the pollutant concentrations dropped to 3. Biosurfactants can also be involved in microbial enhanced oil recovery MEOR. MEOR methods are used to recover oil remaining in reservoirs after primary mechanical and secondary physical recovery procedures [ 61 , 62 ]. It is an important tertiary process where microorganisms or their metabolites, including biosurfactants, biopolymers, biomass, acids, solvents, gases and also enzymes, are used to increase recovery of oil from depleted reservoirs.
Biomedical and therapeutic applications of biosurfactants
Application of biosurfactants in enhanced oil recovery is one of the most promising advanced methods to recover a significant proportion of the residual oil. The remaining oil is often located in regions of the reservoir that are difficult to access and the oil is trapped in the pores by capillary pressure [ 62 ]. This reduces the capillary forces preventing oil from moving through rock pores Figure 4. Biosurfactants can also bind tightly to the oil-water interface and form emulsion.
Bordoloi and Konwar [ 64 ] investigated the recovery of crude oil from a saturated column under laboratory conditions. Laboratory studies on MEOR typically utilize core substrates and columns containing the desired substrate, usually sand. This substrate is used to demonstrate the usefulness of biosurfactants in recovery of oil from reservoirs.
For this purpose, a glass column is packed with dry sand, then the column is saturated with crude oil and aqueous solution of biosurfactant is poured in the column. The potential of biosurfactants in MEOR is estimated by measuring the amount of oil released from the column after pouring the aqueous solution of biosurfactant in the column. Biosurfactants used in the experiment were produced by bacterial isolates of P. The biosurfactant produced by MTCC was reported to be less efficient.
In control samples treated with culture medium, very little recovery of crude oil was obtained. Jinfeng et al. A02 , Pseudomonas sp. P15 and Bacillus sp. The oil production performance in the unit was periodically monitored before, during and after microbial water-flooding and then compared. This situation changed markedly six month later and by the end of the July , about t of additional oil was obtained compared with the predicted oil production.
All the seven production wells showed a positive response to the treatment, of which five wells evidently increased in oil production. Pornsunthorntawee et al. For this purpose, sand-packed column inoculated with a motor oil complex was used. The surfactant solutions were poured onto the packed column to test their ability to enhanced oil recovery. The biosurfactants produced by Bacillus subtilis PT2 could recover oil more effectively than that produced by Pseudomonas aeruginosa SP4. Biosurfactants can also be used to extract hydrocarbon compounds from oil shales in order to utilize it as a substitute for petroleum energy fuel.
In studies conducted by Haddadin et al. Contamination of soil environments with heavy metals is very hazardous for human and other living organisms in the ecosystem. Due to their extremely toxic nature, presence of even low concentrations of heavy metals in the soils has been found to have serious consequences. Nowadays, there are many techniques used to clean up soils contaminated with heavy metals. Remediation of these soils includes non biological methods such as excavation, and disposal of contaminated soil to landfill sites or biological techniques [ 68 ]. Biological methods are processes that use plants phytoremedation or microorganisms bioremediation to remove metals from soil.
Application of microorganisms was discovered many years ago to help in reduction of metal contamination. Heavy metals are not biodegradable; they can only be transferred from one chemical state to another, which changes their mobility and toxicity. Microorganisms can influence metals in several ways. Some forms of metals can be transformed either by redox processes or by alkylation. Metals can also be accumulated by microorganisms by metabolism-independent passive or by intracellular, metabolism-dependent active uptake.
Microorganisms can influence metal mobility indirectly by affecting pH or by producing or releasing substances which change mobility of the metals [ 69 , 70 ]. The first technique used is ex situ —contaminated soil is excavated, put into the glass column and washed with biosurfactant solution. In turn, soil flushing of in situ technologies involves use of drain pipes and trenches for introducing and collecting biosurfactant solution to and from the soil [ 15 , 71 ].
Interestingly, biosurfactants can be used for metal removal from the soil. Biosurfactants can be applied to a small part of contaminated soil in which soil is put in a huge cement mixer, biosurfactant-metal complex is flushed out, soil deposited back, and biosurfactant-metal complex treated to precipitate out biosurfactant, leaving behind the metal.
The bond formed between the positively charged metal and the negatively charged surfactant is so strong that flushing water through soil removes the surfactant metal complex from the soil matrix. This method can also be carried out for deeper subsurface contamination only with more pumping activities. Using biosurfactants have unquestionable advantages because bacterial strains able to produce surface active compounds do not need to have survival ability in heavy metal-contaminated soil. However, using biosurfactants alone requires continuous addition of new portions of these compounds.
The usefulness of biosurfactants for bioremediation of heavy metal contaminated soil is mainly based on their ability to form complexes with metals. The anionic biosurfactants create complexes with metals in a nonionic form by ionic bonds. The cationic biosurfactants can replace the same charged metal ions by competition for some but not all negatively charged surfaces ion exchange. Metal ions can be removed from soil surfaces also by the biosurfactant micelles.
The polar head groups of micelles can bind metals which mobilize the metals in water Figure 5 [ 38 , 71 — 73 ]. Mechanism of biosurfactant activity in metal-contaminated soil [ 74 ]. Biosurfactants which are used in bioremediation of metal-contaminated soils have been proposed for use in metal removal in recent years [ 72 , 73 ]. High potential of biosurfactants in mobilization and decontamination of heavy metal contaminated soil was confirmed by Juwarkar et al.
To study the feasibility of di-rhamnolipid to remove chromium, lead, cadmium and copper from soil, a column study was conducted. Heavy metal spiked soil into a glass column was washed with 0. In turn, Das et al. The positive role of marine biosurfactant in the remediation of polyaromatic hydrocarbons was reported earlier [ 7 ], however there was no information about the role of this biosurfactant in heavy metal remediation. The study revealed that tested anionic biosurfactant was able to bind the metal ions and the percentage removal of Pb and Cd metals varied with the different concentrations of metals and biosurfactants.
The ability of biosurfactant of marine origin to chelate toxic heavy metals and form an insoluble precipitate could be useful in treatment of heavy metal containing wastewater. Removal of heavy metals from sediments could be enhanced by use of solution containing biosurfactant and inorganic compounds. Another effective method for the remediation of heavy metals contaminated soil is biosurfactant foam technology. Wang and Mulligan [ 78 ] evaluated the feasibility of using rhamnolipid foam to remove Cd and Ni from a sandy soil.
They reported that the use of foam had a significant effect on the mobility of biosurfactant flowing in a porous medium and made a more uniform and efficient contact of biosurfactant with the metals. Application of rhamnolipid foam increases efficiency and allows removal of The system used for the experiment is presented schematically by Wang and Mulligan [ 78 ]. The rate of heavy metal removal from soil strongly depends on its chemical composition.
The predominant constituent of the sand and silt fraction in many soils is quartz, thus quartz was chosen for the bioremediation experiment. They observed that the best recovery efficiency from quartz, approximately Biosurfactants were also used to evaluate their potential in arsenic mobilization from the mine tailings [ 79 ]. The experimental results showed that introduction of rhamnolipid enhanced As mobilization from the mine tailings significantly. It has been reported by Doong et al. The high concentration of rhamnolipid required in this experiment could be due to the sorption of biosurfactant to the mine tailings and the dilution and binding effects of mine tailing particles.
The biosurfactant may be enhancing As mobilization by reducing the interfacial tension between As and the mine tailings, by formation of aqueous complexes or micelles and by improving the wettability of the mine tailings. The results from this research study indicated that biosurfactants have potential to be used in the remediation of As-contaminated mine tailings and they can be also effectively used to remove As from soils.
Besides the mobilization, biosurfactants can be involved in other processes connected with remediation of heavy metals. They are used, for example, in entrapping of trivalent chromium in micelles which provides bacterial tolerance and resistance towards high concentration of Cr III. Gnanamani et al. MTCC The first process transforms the toxic state of chromium into less-toxic state and the second process prevents the bacterial cells from the exposure of chromium III.
Both reactions keep bacterial cells active all the time and provide tolerance and resistance toward high hexavalent and trivalent chromium concentrations. Efficiency of phytoremediation of heavy metal contaminated soils can be increased by inoculation of plants by biosurfactant-producing and heavy metal-resistant bacteria.
Biosurfactant-producing Bacillus sp. J strain was investigated for its capability to promote the plant growth and cadmium uptake of rape, maize, sudangrass and tomato in soil contaminated with different levels of Cd [ 82 ]. The study demonstrated that the tested strain could colonize the rhizosphere of all studied plants but its application enhanced biomass and Cd uptake only in plant tissue of tomato. This means that root colonization activity of the introduced strain is plant type influenced. However, further analyses of interactions between the plants and biosurfactant-producing bacterial strain J may provide a new microbe assisted-phytoremediation strategy for metal-polluted soils.
Further work on the applications of biosurfactants and biosurfactants-producing bacteria in phytoremediation, especially in sites co-contaminated with organic and metal pollutants is required. It was estimated by the U. The presence of toxic metals lead, cadmium, arsenic in some cases causes inhibition of organic compound biodegradation [ 83 — 85 ]. These include inoculation with metal-resistant microorganisms, addition of materials like: clay minerals—kaolinite and montmorillonite, calcium carbonate, phosphate, chelating agents EDTA , and bio- and surfactants [ 83 ].
Biosurfactants produced by microorganisms show promise for enhancing organic compound biodegradation in the presence of metals. Application of biosurfactants or microorganism produced biosurfactants in in situ co-contaminated sites bioremediation seems to be more environmentally compatible and more economical than using modified clay complexes or metal chelators. Sandrin et al. This research demonstrated that rhamnolipids induced the release of lipopolisaccharide LPS from gram-negative bacteria, Burkholderia sp.
The authors suggested that rhamnolipid was able to reduce metal toxicity to microbial consortia in co-contaminated soils through a combination of metal complexation and in the alteration of cell surface properties through the release of lipopolisaccharide LPS , resulting in enhanced bioremediation effect. Maslin and Maier [ 85 ] studied the effect of rhamnolipids produced by various Pseudomonas aeruginosa strains on the phenanthrene degradation by indigenous populations in two soils co-contaminated with phenanthrene and cadmium. The authors showed that rhamnolipids applied had the ability to complex cationic metals, increasing the phenanthrene bioavailability [ 85 ].
The biodegradation of phenanthrene was increased from 7. As biosurfactants are degraded by soil populations in 2—3 weeks, Maslin and Maier [ 85 ] used a pulsing strategy, in which new portions of rhamnolipids were added to the system to maintain a constant level of biosurfactant during organic contaminant mineralization. Application of biosurfactant and biosurfactant-producing bacteria in environmental technologies bioremediation and phytoremediation has been studied. Both organic and inorganic contaminants can be removed through different processes physico-chemical and biological in which biosurfactants are involved.
Due to their biodegradability and low toxicity, they are very promising for use in environmental biotechnologies. The commercial success of biosurfactants is still limited by their high production cost. Optimized growth conditions using cheap renewable substrates agro-industrial wastes and novel, efficient methods for isolation and purification of biosurfactants could make their production more economically feasible.
Another important aspect regarding biological remediation technologies is the use of biosurfactant in the process on a large scale. To felicitate this process, new techniques should be developed such as foams or micro-foams colloidal gas aphrons-CGA in conjunction with biosurfactants. Little is known about the potential of biosurfactant production by microorganisms in situ.
Most of the described studies were done under laboratory conditions. More efforts are required to evaluate the biosurfactant production by microorganisms in situ and their role in biological remediation technologies. Remediation systems with only one type of the contaminant have been studied to gain a basic understanding. Only a few studies have also been completed on metal-organic pollutant co-contaminated site remediation [ 86 ].
More information is required concerning the structures of biosurfactants, their interaction with soil and contaminants and scale up and cost effective biosurfactant production [ 86 ]. For lowering the cost of biosurfactant production, commercially viable biological and engineering solutions are required. One important point in this context is the use of low cost substrates for production of biosurfactants. A promising approach seems to be the application of inoculants of biosurfactant producing bacteria in phytoremediation of hydrocarbon polluted soil to improve the efficiency of this technology.
Application of the biosurfactants in phytoremediation on a large scale requires studies to identify their potential toxic effect on plants. Mixture of different surfactants often presents better properties than the individual surfactants, due to synergistic effect. An improved strategy to surfactant-enhanced remediation SER is to apply mixture of surfactants at reduced concentration of individual surfactants, which reduces the cost while maintaining the efficiency of remediation [ 17 ].
Solubilization of hydrophobic contaminants is improved by using mixtures of anionic and nonionic surfactants, which was shown to exhibit synergistic interaction, suggesting that appropriate combinations of surfactants have the potential to enhance the efficiency of soil washing and flushing and to facilitate the bioavailability of pollutants [ 28 , 29 ]. Trehalose lipids produced by Rhodococcus erythropolis were shown to have good solubilization capacity for hydrophobic compounds such as phenanthrene, and great potential for applications in bioremediation of sites contaminated with PAHs [ 30 ].
Combining Tween 80, Triton X and biosurfactants from P. Interesting, degradation of phenanthrene was completely inhibited for all the surfactants tested at concentration higher that their CMC, suggesting that the combination of surfactant and biosurfactant has potential in bioremediation however it requires a research in a case-by-case basis. The phenanthrene-degrading strain B-UM lacks the ability to produce surfactants for dissolution of phenanthrene, and the direct adhesion of cells to phenanthrene surface might be the major pathway for B-UM to take up this PAH [ 33 ].
As expected, addition of surfactants inhibited the phenanthrene degradation by B-UM, and similar effect were found with addition of Triton X to Arthrobacte r sp. Kumar et al. It was shown that this strain is capable of utilizing up to four-ring PAHs but not hexadecane and octadecane as a sole carbon and energy source, and the authors identified the presence of both tension-active and emulsifying activities, suggesting that IR1 produces biosurfactants on both water miscible and immiscible substrates. Rhamnolipids together with anthracene-degrading bacteria had a dramatic increase in the solubility of anthracene by the bacterium strains and, interesting, it was observed the metabolism of biosurfactant by one of the strains which appears to be an important event on this process [ 36 ].
Addition of Tween 60, nonionic surfactant, to a Rhodococcus rhodochrous strain in liquid media was also shown to improve biodegradation of fluorene by being an additional carbon source to the bacterial cells [ 37 ]. Moreover, the combination of biostimulation and bioaugmentation was shown to result in significant removal of phenanthrene under Antarctic environmental conditions. The authors combined a complex organic source of nutrients fish meal , a surfactant Brij and a psychrotolerant PAH-degrading bacterial consortium and it was shown that the combined treatment is more efficient than the biostimulation or bioaugmentation isolated [ 38 ].
Indigenous or injected biosurfactant-producing microorganisms are exploited in oil recovery in oil-producing wells. Microbial enhanced oil recovery MEOR is often implemented by direct injection of nutrients with microbes that are able of producing desired products for mobilization of oil, by injection of a consortium or specific microorganisms or by injection of the purified microbial products e.
However, application of MEOR requires a thoroughly research on a case-by-case basis taking in account the physical-chemical conditions and soil and rock formation characteristics. The characteristics of the oil that has been already recovered from the well will also impact the MEOR application. MEOR is a powerful technique to recover oil, especially from reservoirs with low permeability or crude oil with high viscosity, but the uncertainties on the results and costs are a major barrier to its widespread.
Oilfield emulsions are formed at various stages of petroleum exploration, production and oil recovery and processing, and represent one of the major problems for the petroleum industry [ 39 ], which requires a de-emulsification process in order to recover oil from theses emulsions. Biosurfactants have the potential to replace the use of chemical de-emulsifiers in situ, saving on transport of the oil emulsion and providing a more environmentally-friendly solution.
Among the bacteria species, Acinetobacter and Pseudomonas species are the main de-emulsifiers in the mixed cultures [ 40 ]. Glycolipids e.
The major advantage of using microorganisms or their products over chemical products is the disposal of the de-emulsifier in the aqueous phase and its removal from the oil phase, since emulsion formation is required in further processing steps. Microbes and biosurfactants are in general readily biodegradable, which allows a cheap and easy removal of the de-emulsifier after this process. Surfactants have potential application for oil recovery from petroleum tank bottom sludges and facilitating heavy crude transport through pipelines. It was shown that rhamnolipids can be used to remove soaked oil from the used oil sorbents [ 30 ].
Petroleum hydrocarbons are an essential raw material in our current society, but they also constitute a major environmental pollutant that is very difficult to be bioremediated. Crude oils have very low water solubility, high adsorption onto soil matrix and present limited rate of mass transfer for biodegradation [ 2 ]. Oil-contaminated soil is especially difficult for bioremediation since oil excess forms droplets or films on soil particles, which is a powerful barrier against microbial degradation [ 41 ]. Bioavailability of contaminants in soil to the metabolizing organisms is influenced by factors such as desorption, diffusion and dissolution.
Biosurfactants are produced to decrease the tension at the hydrocarbon-water interface aiming to pseudosolubilize the hydrocarbons, thus increasing mobility, bioavailability and consequent biodegradation [ 42 ]. Several biosurfactant are produced by a diversity of microorganisms in order to survive in an oil-rich environment, and this adaptation process selected for surfactants with highly adaptable phycal-chemical properties. Biosurfactants are, therefore, very suitable for applications in the oil industry and this is reflected in the market, where the large majority of biosurfactants produced are in petroleum-related applications [ 21 ].
The applications are, in general, in oil recovery, oil spill management, MEOR and as oil dispersants and demulsifiers [ 18 ]. Purified rhamnolipid biosurfactants were applied in the removal of oil from contaminated sandy soil [ 22 ]. The authors optimized the biosurfactant and oil concentrations in the removal of oil applying statistical experimental design tool that generates a surface response. Rhamnolipids, when added above CMC, enhanced the apparent aqueous solubility of hexadecane, the biodegradation of hexadecane, n-paraffins, octadecane, creosotes in soil and promoted biodegradation of petroleum sludges [ 43 , 44 ].
Rhamnolipids produced by Nocardioides sp. A-8 allows this bacterium to grown on aromatic hydrocarbons or n-paraffin as sole carbon source by lowering the surface tension and emulsifying the aromatic hydrocarbons [ 45 ]. The authors found similar results for the strain Pantoea sp. A, which also produces rhamnolipid to grow on n-paraffin or kerosene. Both A-8 and A strains were isolated together with other 15 aerobic microbial isolates from oil-contaminated sites in Antarctica and appear to be very promising source for application in accelerated environmental bioremediation at low temperatures.
Urum and Pekdemir [ 41 ] applied different biosurfactants rhamnolipid, aescin, saponin, lecithin and tannin in washing oil-contaminated soil and observed significant removal of crude oil with different concentrations of biosurfactant solution. Oil mobilization was the main cause for its removal, which was triggered by the reduction of surface and interfacial tensions, rather than oil solubilization or emulsification. This work was followed by a comparison of a biosurfactant, rhamnolipid, and a chemical surfactant, SDS, in removal of crude oil from soil [ 46 ].
Urum et al [ 47 ] then compared the efficiency on oil removal from soil using rhamnolipid and saponin biosurfactants and SDS, and the results showed a preferential removal of oil by the surfactants. SDS was more effective for aliphatic than aromatic hydrocarbons, whereas biosurfactants removed more aromatics.
These results provide insights on the formulation of surfactant combinations, suggesting that the strategy should consider the degree of aromaticity in the crude oil-contaminated soil. The combination of oil-degrading bacteria and biosurfactant or biosurfactant-producing bacteria has also been tested by research groups. This treatment resulted in increased oil emulsification and also adhesion of hydrocarbon to the bacteria cell surface.
Rhamnolipids have potential microbial activity. It has been shown that these biosurfactants are very efficient bacteriostatic agent against Listeria monocytogenes, an important food-related pathogen, and showed synergistic effect when combined with nisin, a broad-spectrum bacteriocin [ 48 ]. Both biosurfactants and surfactin were shown to be able to reduce bacterial adhesion to polystyrene surfaces more efficient than the chemical surfactant sodium dodecyl sulfate. Moreover, rhamnolipids were shown to significantly reduce the rate of deposition and adhesion, in rinsed chamber with these biosurfactants, of several bacterial and yeast strains isolated from explanted voice prostheses [ 50 ].
Rhamnolipids are also known to remove heavy metals. MEL, a glycolipid, is a potent antimicrobial agent, especially against gram-positive bacteria. MEL has also been shown to induce growth arrest, apoptosis and differentiation of mouse malignant melanoma cell cultures [ 52 , 53 ].
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MEL-A produced by Pseudozyma antartica T has been shown to have cell differentiation activities against human leukemia cells, mouse melanoma and PC12 cells [ 54 ]. Delivery of siRNA into the cytoplasm is still a challenge and a barrier for more gene silencing-based individualized drugs. MEL-A-containing cationic liposomes has been shown to directly deliver siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway with better results compared with viral vectors in clinical applications [ 55 ].
Lipopeptides, such as surfactins, are particularly interesting because of their high surface activities and antibiotic potential. These biosurfactants have been reported as antibiotics, antiviral and antitumor agents, also as immunomodulators and inhibitors of specific toxins and enzymes. Surfactin, a cyclic lipopeptide, is known to be active in several biological activities, such as induction of ion channel formation, antiviral and antitumor, anti-inflammatory agent reviewed by Rodrigues et al.
Moreover, pre-coated vinyl urethral catheters with surfactin were shown to reduce biofilm formation by several Salmonella species and other infectious bacteria [ 56 ]. Surfactin has been shown to be more efficient than chemical surfactants sodium dodecyl sulphate or dodecylamine in improving floatability of metal-laden sorbents under similar conditions [ 57 ].
Also, surfactin was shown to contribute to reduce colonization of pathogenic bacteria, such as L. Biosurfactants are considered ideal for environmental application, due to their numerous advantages over their chemical counterparts, such as biodegradability and less toxicity [ 60 ]. Among the biosurfactants, rhamnolipids are the better studied and promising candidates for large scale production especially because of their notable tensoative and emulsifying properties [ 61 - 63 ].
Besides these, twenty-five rhamnolipid congeners have been described in P. Free HAAs also show surface-tension activities and have been directly related to the promotion of swarming motility [ 10 ]. Rhamnose sugar is widely found in bacteria and plants, but not in humans. The activated L-rhamnose is derived from a glucose scaffold in four sequential steps, yielding deoxy-thymidine di-phospho dTDP -L-rhamnose.
The catalytic activity of RmlA is allosterically regulated by the final product of the pathway, dTDP-L-rhamnose [ 67 ], which makes RmlA the regulatory sensor for the downstream pathway. RmlA is a homotetramer with the monomer consisting of three functional domains: one core domain that shares the sequence similarity with nucleotidyltransferases, and two other domains that contain the recognition and binding sites for the nucleotide and sugarphosphate [ 67 ].
All four enzyme genes are organized as a single operon in P. Apparently, the key regulatory targets for rhamnolipid production in P. The rhlAB operon is transcriptionally and posttranscriptionally regulated by manifold factors, commonly associated to the quorum sensing system, some of which also participates of the regulation of rmlBDAC operon. The quorum sensing QS system is a bacterial communication system characterized by the secretion and detection of signal molecules — autoinducers — within a bacterial population. QS is a global regulatory system found in most bacterial species controlling several and diverse biological functions, such as virulence, biofilm formation, bioluminescence and bacterial conjugation [ 70 ].
The main components of a quorum sensing system are the QS signal synthase, the signal receptor regulatory protein , and the signal molecule [ 71 ]. There are two known conventional QS systems in P. There has been postulated that sequences conserved in some promoters regions of RhlR and LasR-regulated genes are responsible by these regulation [ 75 ]. It can be verified that some QS-regulated genes belong specifically to rhl regulon, and some sequences RhlR-specific can be determined in promoters regions of some genes regulated by rhl system [ 76 ]. Although the rhl system has already been considered las -dependent [ 77 ], it has been shown that the expression of rhl system is maintained in a lasR mutant [ 72 ].
Rhamnolipid production in P. The rhlR gene is known to have four different transcription start sites [ 78 ]. In addition, in the last few years many other factors related to the QS system have been identified and reported to directly or indirectly influence rhamnolipid production. Stationary phase is a physiological state frequently related to nutrient scarcity. Rhamnolipids is also associated with physiological roles in the uptake of poorly accessible substrates and is often associated with bacterial response to nutrient-deficient environments. The production is repressed in exponential phase and low cell density while is activated in stationary phase and high cell densities.
This environmental regulation is related to several factors that directly or indirectly control transcription and post-transcription levels of expression. The production in stationary phase is related to factors which contribute to the production in this phase and with other factors that inhibit the production in the exponential phase. The main factor that contributes to the rhamnolipid production in stationary phase appears to be the rhl system.
This QS system is activated in high cell densities, so it represents a direct link between high cell population and rhamnolipid production activation. The second factor that guarantees the rhamnolipid production on stationary phase is related to a posttranscriptional regulation. It has been shown that most QS-regulated genes are not induced before the stationary growth phase, even when exogenous acyl-HSL signals are present in early growth phases [ 81 ]. These findings clearly indicate the other factors are involved in the expression of QS-regulated genes. In gidA mutants, the levels of rhlR mRNA are similar to the levels in wild-type, whereas rhlA mRNA levels were significantly decreased, suggesting that the rhamnolipid production is controlled via posttranscriptional modulation of RhlR levels by GidA [ 82 ].
In relation to factors that inhibit the rhamnolipid production in the exponential phase, two regulators can be identified. The first of them is the QscR factor. It has been demonstrated that the rhlAB operon is present in the subset of genes repressed by QscR in the exponential phase [ 83 ]. This factor is a luxR-homologue protein that integrates the QS regulatory network and controls a distinct but overlapping regulon with the las and rhl systems [ 83 ].
This can explain the effect of the QscR over the rhlAB operon. The second regulator is the DksA protein. DksA synthesis reaches its maximum during the exponential growth phase and it is posttranscriptionally downregulated during the late exponential and stationary growth phase [ 86 ]. Beyond the factors related to the growth phase, an important factor related to the QS systems which influences rhamnolipid production in P. PQS is involved in a complex regulatory network of QS connecting systems las and rhl.
PqsR is activated by las QS system and repressed by rhl QS system, evidencing a complex network of regulation [ 89 ]. PQS was shown to be related to rhamnolipid production in different ways. Firstly, PQS production occurs in the late logarithmic phase and reaches its maximum in the late stationary phase [ 87 , 88 ], with a similar profile to that of rhamnolipid biosynthesis. It was shown that pqsR and pqsE mutants have reduced rhamnolipid production, even when exposed to wild-type levels of C4-HSL [ 78 ].
Also, it has been hypothesized that PqsR is essential for rhamnolipid production, since PQS does not overcome the absence of pqsR in the regulation of phz1 operon, also involved in rhl -dependent phenotype [ 74 ]. As PqsR controls PQS production and rhamnolipid production is abolished in the absence of PQS signaling [ 87 ], it is possible that this effect is indirect. An important environmental condition which highly influences the rhamnolipid production in P. Conditions such as nutrient deprivation and nitrogen exhaustion, even in a QS-independent manner, contribute to an increase in rhamnolipid production.
Thus, several regulatory factors have been identified in last years which connect this condition to specific gene regulation patterns. The sigma factor of stationary phase, RpoS, plays an important role in the response to different stress conditions in P. RpoS levels increase at the onset of stationary phase and in response to nutrient deprivation, even at low cell densities [ 91 ].
RpoS regulon comprises virtually all genes regulated in stationary phase, and has several overlaps with las and rhl regulons [ 92 ]. RpoS is involved in rhamnolipid production by two different manners, which indicates a genetic link between rhamnolipid production and nutrient deprivation and environment stress adaptation. Firstly, the rhlAB operon integrates the RpoS regulon and has been shown to be upregulated and partially RpoS-dependent [ 93 ].
Furthermore, RpoS was shown to be required for swarming motility [ 94 ], a phenotype related to HAAs and rhamnolipids [ 10 ]. The rhamnolipid production is largely dependent on nitrogen exhaustion in P. Moreover, nitrate has been shown to provide the highest yields of rhamnolipid production [ 7 ], related to upregulation of glutamine synthase under nitrogen-limiting conditions by RpoN [ 97 ].
On the other hand, the activation of rpoN-dependent promoters seems to be CbrB and NtrC-dependent [ 99 ]. The rhlAB operon and rhlR are controlled indirectly and directly, respectively, by RpoN [ 93 ], since under nitrogen-limiting conditions, P. Another important factor that can correlate rhamnolipid production to stress conditions, but also with quorum sensing, is the PQS. PQS is involved in stress response in P. This clearly indicates the interconnection between rhamnolipid and PQS synthesis.
The great adaptability of P. Different regulatory factors that control virulence in P. Vfr, the global regulator of the virulence in P. It affects the expression of multiple virulence factors downstream in the QS cascade, including rhamnolipid production. Another factor related to virulence is the VqsR protein.
This is a LuxR homologue that has been shown to modulate the expression of QS genes and others metabolic process, seeming to be a global regulator in P. Rhamnolipid production is reduced in vqsR mutants, which can be related to the influence of this factor in the QS system, since most of the VqsR-regulated genes were previously identified as QS-regulated [ 99 , ]. PtxR, a LysR-type transcriptional regulator, which modulates the production of virulence factors in P.
The rhamnolipid synthesis increase in ptxR mutants, which can be related to its modulation of the QS system, since the ptxR mutants showed upregulation of rhlI and lasI genes, as well as the C4-HSL and 3OCHSL autoinducers, when compared to the wild type strain [ ]. Biofilm formation provides several advantages to the bacteria, providing protection that may enhance bacterial survival under environmental stress conditions. Rhamnolipids play a major role in the architecture of biofilms produced by P. The cell detachment of the biofilm structure and the formation of water channels have been shown to be dependent of the rhamnolipids synthesis.
It has been reported that rhamnolipid production is related to biofilms in P. AlgR was shown to be the main repressor of rhamnolipid production within adherent biofilms and during its development, acting as a repressor of the expression of rhlI and rhlAB [ ]. No such effect was reported on planktonic growth so far, therefore, it was hypothesized that AlgR acts through a contact-dependent or biofilm-specific mode of regulation.
BqsS-BqsR, a two component system, has also been reported to be related to biofilm formation in P. Although the environmental stimuli that trigger BqsS-BqsR activity are still unknown, evidences support the existence of opposite effects of AlgR and BqsS-BqsR on rhamnolipid production in the context of biofilm formation [ ].
Besides the known environmental stimuli, others are likely to play important role in rhamnolipid production. Some regulatory factors have been identified as involved on rhamnolipids production, however their environmental stimuli are still to be discovered. GacS-GacA is a well characterized two-component system in P. RsmA is a translational regulator and acts by preventing the translation initiation of target RNAs.
However, RsmA also activates indirectly gene expression, by acting over repressor factors. GacS-GacA can have opposite effects on rhamnolipids production, evidencing a complex regulation. Another global regulator involved in rhamnolipids production is VqsM, an AraC-type transcriptional regulator. VqsM has been shown to modulate the expression of several genes, including QS regulators. The environmental stimuli for this regulation are also unknown.
Several microorganisms are known to produce biosurfactants that can vary in structure and chemical composition. These variations are dependent on the producing microorganism, raw matter used for fermentation and conditions of fermentative process [ 17 ]. According to recent data, global biosurfactants market was worth USD 1,7 bi in and is expected to reach USD 2,2 bi in , based on a growth rate of 3. The global biosurfactants market volume is expected to reach , The number of publications related to identification, optimization of production process and better understanding of the metabolic pathways has increased in recent years [ 5 ].
Many biosurfactants and their production processes have been patented, but only some of them have been commercialized [ 6 ]. Some examples of products based on biosurfactants that are available in the market are shown in Table 2. Besides the research efforts, the cost for biosurfactant production is approximately three to ten times more than the cost to produce a chemical surfactant. Biosurfactants are typically produced by microorganisms growing in hydrocarbons as a carbon source, which are usually expensive increasing the production cost [ 6 ]. In addition, the downstream cost, low productivity and intense foaming formation during the biosurfactant production currently is a barrier for an economically viable production of biosurfactant [ 3 , 6 ].
Therefore, most researches have been focusing on increasing the production yield, reducing raw material cost and developing oxygenation strategies to reduce foaming formation [ 4 , 6 , 61 ]. Several approaches have been applied in order to improve biosurfactant productivity such as optimization of growth conditions e.
Rhamnolipids are mainly produced by Pseudomonas species, such as Pseudomonas aeruginosa. This bacterium produces rhamnolipids as secondary metabolite and their production coincides with the stationary growth phase [ 65 , 95 , ]. Rhamnolipids can be produced using varies carbon sources, such as vegetable oils e. The carbon and nitrogen sources are important factors in the production of these biosurfactants and have great influence their production cost and considerable efforts have been done towards the use of agro-industrial byproducts and renewable resources as substrates in the production process [ 3 ].
Studies have been shown that use of inexpensive substrates, such as crude or waste materials, dramatically affects the production costs of biosurfactants [ 3 ]. Different waste substrates have been used for rhamnolipid production, such as fatty acids from soybean oil refinery wastes [ ], glycerin from biodiesel production waste [ ] and sunflower-oil refinery waste [ ]. Nevertheless, the potential of rhamnolipids production from renewable resources is so far not fully exploited. According to Henkel et al [ 3 ], use of waste substrates in the production processes is likely to increase its influence on the field of microorganism-based production, since they are usually cheaper, maximize the utilization efficiency regarding the overall production process and makes the process more environmentally friendly.
Currently, the main nitrogen source used to promote rhamnolipid production is nitrate [ 5 ]. Interestingly, in contrast with nitrate, ammonium has been shown to prevent rhamnolipid production [ - ]. Growth limiting conditions are known to promote rhamnolipids production, as well as other secondary metabolites.
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