Synthesis of rhamnolipid biosurfactant and mode of hexadecane uptake by Pseudomonas species
© Cameotra and Singh; licensee BioMed Central Ltd. 2009
Received: 17 November 2008
Accepted: 11 March 2009
Published: 11 March 2009
Microorganisms have devised ways by which they increase the bioavailability of many water immiscible substrates whose degradation rates are limited by their low water solubility. Hexadecane is one such water immiscible hydrocarbon substrate which forms an important constituent of oil. One major mechanism employed by hydrocarbon degrading organisms to utilize such substrates is the production of biosurfactants. However, much of the overall mechanism by which such organisms utilize hydrocarbon substrate still remains a mystery.
With an aim to gain more insight into hydrocarbon uptake mechanism, an efficient biosurfactant producing and n-hexadecane utilizing Pseudomonas sp was isolated from oil contaminated soil which was found to produce rhamnolipid type of biosurfactant containing a total of 13 congeners. Biosurfactant action brought about the dispersion of hexadecane to droplets smaller than 0.22 μm increasing the availability of the hydrocarbon to the degrading organism. Involvement of biosurfactant was further confirmed by electron microscopic studies. Biosurfactant formed an emulsion with hexadecane thereby facilitating increased contact between hydrocarbon and the degrading bacteria. Interestingly, it was observed that "internalization" of "biosurfactant layered hydrocarbon droplet" was taking place suggesting a mechanism similar in appearance to active pinocytosis, a fact not earlier visually reported in bacterial systems for hydrocarbon uptake.
This study throws more light on the uptake mechanism of hydrocarbon by Pseudomonas aeruginosa. We report here a new and exciting line of research for hydrocarbon uptake involving internalization of biosurfactant covered hydrocarbon inside cell for subsequent breakdown.
Pollution due to petroleum oil is even now a prevalent ecological hazard and hence microbial degradation of hydrocarbons remains as topical an issue as before. A common feature of all the fractions of crude oil is their low water solubility and this poses special problems for those microorganisms capable of utilizing such water immiscible substrates as source of carbon and energy. The first step in the process is the transport of the hydrocarbon from oil phase to the cell surface in some way so as to achieve effective cell surface contact and ultimately efficient transportation across cell membrane, or in other words uptake/intake. Although much work has been done in this field, the mechanism of n-alkane transport to bacterial cell and its subsequent assimilation inside cells still remains obscure.
Two types of hydrocarbon interaction during biodegradation has been described earlier by Kirschner : adhesion to oil, and a hypothesized pseudosolubilization in which the hydrocarbon degrading bacteria assimilate small droplets of emulsified oil. Reports are available in favor of the first theory where microbial cells attach to the surface of hydrocarbon drops much smaller than the cells and substrate uptake presumably takes place through diffusion or active transport at the point of contact [2, 3]. The hypothesized emulsification and incorporation of submicron droplets of oil [4, 5] is analogous to the pseudosolubilization of oil hypothesized by Kirschner. Similar theory was explained by Singh and Desai . They referred to two modes of initial interaction of hydrocarbon with the microbial cells: (a) Direct contact of microorganisms with insoluble substrate (unmediated interaction) or (b) by the contact through a mediator (mediated interaction. The scale tilts in the favor of the latter theory involving the role of mediators or extracellular solubilizing factor.
Several investigators have shown the presence of emulsifiers in the culture broth during the growth of microorganisms on hydrocarbons and its effect, due to solubilization/emulsification, on hydrocarbon uptake [7–9]. Biosurfactants are present in various pools inside cells: as intracellular molecules, extracellularly secreted compounds or as compounds located at the cell surface . Together they have been known to enhance degradation by alteration in cell hydrophobicity and enhancement of dispersion of water immiscible compounds [11–13]. P. aeruginosa strains are known to produce rhamnolipid type of biosurfactant, reported to be a mixture of mainly mono and dirhamnolipids. Addition of rhamnolipids to pure cultures or in soil systems has been shown to enhance biodegradation of a number of hydrocarbons including hexadecane [14–19]. Thus a clear correlation exists between surface active agent production and alkane utilization by the degrading organism. However, Different modes of uptake have been proposed for different microorganisms for the growth on hydrocarbons, sometimes more than one mechanism occurring simultaneously . Hence, no conclusive picture has come out of the various studies till now.
In the present study, we have tried to look into the matter of hydrocarbon uptake by a hydrocarbon degrading Pseudomonas strain isolated from oil-contaminated soil. Characterization of biosurfactant produced was done and its role in hydrocarbon uptake studied. Electron Microscopic studies were done extensively to elucidate the mechanism(s) adopted to uptake water insoluble hydrocarbon into bacterial cell and some interesting results were obtained which would go on to consolidate the theories of hydrocarbon uptake by bacterial cells.
Chemicals and media
All the chemicals used were of the highest purity grade. Minimal media used in the study contained 28 mM disodium hydrogen phosphate, 14.7 mM potassium dihydrogen orthophosphate, 6.1 mM ammonium sulphate, 6.6 mM magnesium sulphate and 1ml of trace element solution (TES) per liter. TES contained 1.30 mM Al(OH)3, 0.22 mM SnCl2.2H2O, 0.30 mM KI, 1.2 mM LiCl, 0.42 mM MgSO4.4H2O, 8.10 mM H3BO3, 0.35 mM ZnSO4.7H2O, 0.42 mM CoCl2.6H2O, 0.38 mM NiSO4.6H2O, 0.24 mM BaCl2 and 0.35 mM (NH4)I. n-hexadecane used as the test hydrocarbon was obtained from Sigma.
Isolation and biodegradation studies
The organism used in the study was isolated from soil contaminated with crude oil sludge. In all the growth studies, temperature of incubation was 37°C and aeration was maintained by agitation on a rotary shaker (210 rpm). A bacterial isolate designated as SSC2 was isolated from oil contaminated soil by serial dilution plating method on Nutrient Agar media as well as on minimal medium with 2% (v/v) n-hexadecane as the sole source of carbon and energy. Screening was done by (i) monitoring change in dry biomass upon different times of growth on n- hexadecane and (ii) residual hydrocarbon (n-hexadecane) analysis. Degradation of n-hexadecane was monitored by Gas-Chromatographic analysis of residual hydrocarbon extracted using hexane as the solvent. For GC analysis, a Hewlett Packard (series II Gas 5890) instrument with cross-linked methyl silicone gum capillary column of 25 mm × 0.2 mm × 0.33 μm film thickness and equipped with FID was used. Oven temperature was standardized at 300°C with injector and detector at 350°C.
Characterization of the selected isolate
SSC2 was characterized using various morphological and biochemical tests and confirmed by whole cell fatty acid profile analysis by GC- Microbial Identification System (MIS) procured from Hewlett Packard. For identification by morphological and biochemical means, Bergy's Manual of Systemic Bacteriology, Vol. 1 and the Manual for the Identification of Medical Bacteria by Cowan & Steel (Second Edition, Cambridge University press) was used. Final confirmation by 16srDNA sequence analyses was also done.
SSC2 was studied for the production of biosurfactant by monitoring the lowering of surface tension of the cell free broth after different time intervals. Surface Tension was measured using a DuNouy Tensiometer (CSC NO. 70535, CSC, Albany, NY, USA) and surface tension of broth was directly recorded in dynes cm-1. Cell free broth of different day's growth was used to study the presence of different sized hydrocarbon droplets in the culture media. SSC2 was grown in minimal media with n-hexadecane as the sole carbon source. Cell free broth was filtered through 0.45 and 0.22 μm Millipore membrane filters. Residual hexadecane was extracted from the filtrates using hexane and analyzed by GC using the same parameters mentioned earlier. Another bacterial soil isolate SSC1, a not so efficient hydrocarbon degrader and biosurfactant producer, was also processed in similar way for studying the formation of varying sized droplets. Another unfiltered parallel set for both the strains was used for total residual hydrocarbon analyses at different times and used for percent hydrocarbon calculation after filtration.
Electron Microscopy studies for hydrocarbon uptake
Scanning Electron Microscopy (SEM) was performed on SSC2 grown in minimal medium with 2% n-hexadecane as carbon source. After 2, 4 and 6 days of growth, cells were spinned down and washed thrice with PBS (phosphate buffer saline, pH 7.2) before being fixed in 2.5% glutaraldehyde made in PBS with overnight incubation at 4°C. After three successive gentle washings in PBS, cells were suspended in minimum amount of PBS and layered onto 1mg/ml polylysine coated cover slips. These were then dehydrated through a series of alcohol dehydration steps (30, 50, 70, 90 and 100%) and finally layered with t-butyl alcohol for freeze-drying. Freeze dried samples were viewed under a Jeol Scanning Electron Microscope-6100. Cells grown for 48–60 hours on 2% (w/v) glucose in minimal medium were taken as control and processed similarly.
For ultrastructural studies, 2, 4 and 6 day old samples were washed in 10 mM phosphate buffer (pH7.2), fixed in 2.5% gluteraldehyde and post fixed in 1% osmium tetraoxide after successive washings in 10 mM sodium phosphate buffer pH 7.4. After several washes in the same buffer, cells were dehydrated in graded acetone solutions and embedded in CY 212 araldite, along with Dodecenyl Succinic Anhydride (DDSA) as hardner, and tri(dimethylaminomethyl) phenol (DMP-30) as catalyst. Ultrathin sections of 60–80 nm thickness were cut using an ultracut E (Reichert Jung) ultramicrotome and the sections were stained in alcoholic uranyl acetate (10 min) and subsequently in lead acetate (10 min) before examining the grids in either CM-10 Phillips Transmission Electron Microscope (TEM) operated at 80kV or Morgagni 268 D Transmission Electron Microscope FEI operated at 80kV which gave better resolution at low magnification. Cells grown for 48–60 hours on 2% (w/v) glucose in minimal medium were taken as control and processed similarly.
Analyses of intracellular hydrocarbon
To analyze the nature of numerous intracytoplasmic inclusions observed in hydrocarbon grown cells 2, 3 and 5 day n-hexadecane grown cells were harvested and washed with phosphate buffer (10 mM; pH 7.0) and with 50 mM tris (hydroxymethyl) aminomethane chloride buffer (pH 7.8) to remove all the adhering extracellular hydrocarbons . 2 and 3 day old SSC2 cells grown on 2% glucose were taken as control and processed similarly. Intracytoplasmic hydrocarbon extraction was done by chloroform using lyophilized cells . 2 μl of the extracted organic phase was analyzed using Shimadzu GC-17A equipped with Flame Ionization Detector and a DB1 column 30 m × 0.25 mm in size. Injector temperature was 250°C with the detector at 300°C. Oven temperature was kept at 100°C for 2 min and increased to 300°C at a rate of 20°C per min. and kept at 300°C for 20 min.
Results and discussion
Isolation and characterization of the hydrocarbon degrading strain
Production of biosurfactant and pseudosolubilization of hydrocarbons
Electron Microscopic studies
In the present study, many mechanisms proposed for hydrocarbon uptake were found to be playing a joint role in facilitating n-hexadecane uptake:
Direct contact of cells with large hydrocarbon droplets.
Interaction of cells with "solubilized/pseudosolubilized" or "accommodated" hydrocarbon droplets of size comparable to the cell size (enhanced dispersion and pseudosolubilization of hydrophobic substrate being the process facilitating enhanced contact for uptake of the substrate)
Uptake of hydrocarbon by internalization of biosurfactant covered substrate.
A process similar in appearance to active pinocytosis was observed in the present electron microscopic studies, for the internalization of biosurfactant covered hydrocarbon droplets of large sizes. Earlier this theory has been proposed in yeasts where vesicle formation at the cell surface was suggested to arise by pinocytosis of alkane . No occurrence/report of a similar process is there for bacteria. In one of the closest studies on different mechanisms of hydrocarbon uptake by bacteria, it was proposed that biosurfactant only act to enhance the process of hexadecane uptake and are not required for the process to take place . However, our electron microscopic findings question this hypothesis and we propose the involvement of biosurfactant to be extended beyond just enhancement of the bioavailability of the substrate. The mechanism reported here is similar to the one proposed by Ratledge  whereby surfactants act to enhance the process of pseudosolubilization/emulsification thereby increasing the availability of hydrocarbon to cells. However, the role of biosurfactant does not end here. Hydrocarbon droplet enclosed within a surfactant layer is the form in which cells seem to take up the hydrocarbon. As proposed earlier by Ratledge , the requirement of energy for the uptake process would be divided between pinocytosis and movement of the alkane free surfactant from inside the cell (through the cell envelope) to the outside. Another factor could be utilization of the internalized biosurfactant for the formation of membranes around the hydrocarbon droplet and its further partitioning into smaller compartments for increased surface area for metabolism. The entire process however, is a combination of predominantly two modes of uptake proposed earlier viz. uptake by direct contact and pseudosolubilization of hydrocarbon to microdroplets, both facilitated by biosurfactants and occurring simultaneously to enhance and facilitate hexadecane uptake.
This paper is dedicated to Mr. Darshan Singh Cameotra, father of SSC who was a source of inspiration and support. We are grateful for the facilities provided for this work to the Director IMTECH, Chandigarh. We acknowledge the help received from AIIMS, New Delhi for the EM studies.
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