Structural and physico-chemical characterization of a dirhamnolipid biosurfactant purified from Pseudomonas aeruginosa: application of crude biosurfactant in enhanced oil recovery

Abstract

The present study describes the structural characterization and biotechnological application of a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa strain NBTU-01 isolated from a petroleum oil-contaminated soil sample. Characterization of partially purified biosurfactant by LC-MS/MS analysis indicated predominant production (78%) of dirhamnolipids Rha-Rha-C10-C10 and Rha-Rha-C12-C10 with a minor production of monorhamnolipids (22%). NMR analysis of the purified major biosurfactant produced by P. aeruginosa strain NBTU-01 identified it as dirhamnolipid and its deduced structure was L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate. The LC-MS/MS analysis of intracellular proteins of P. aeruginosa strain NBTU-01 identified key enzymes and other proteins associated with regulation and biosynthesis of rhamnolipids. Critical micelle concentration of purified dirhamnolipid biosurfactant was determined at 72 ​± 2.25 mg l⁻¹ and it reduced the surface tension of water from 72.2 to 29.5 mN m⁻¹. The crude rhamnolipid biosurfactant effectively emulsified crude petroleum-oil, diesel, kerosene and coconut oil, and removed 70 ± 3.5% crude petroleum oil from a saturated sand pack column. Heating the crude rhamnolipid biosurfactant at 100 °C for 5 h did not affect oil recovery from the sand pack column. Moreover, rhamnolipid biosurfactant showed stability at pH values between 6.0 and 10.0. The crystallization and melting temperature of purified dirhamnolipid biosurfactant was found to be 99 and 134 °C, respectively, suggesting it can withstand thermal denaturation and is suitable for application in high temperature wells for tertiary oil recovery.

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Introduction

Biosurfactants are structurally diverse groups of surface active amphiphilic molecules comprised of different chemical structures such as glycolipids, lipopeptides, polysaccharide–protein complexes, protein-like substances, lipopolysaccharides, phospholipids, fatty acids and neutral lipids.^1–7 Therefore, it is reasonable to expect diverse properties and physiological functions for different groups of biosurfactants. Among the different classes of biosurfactants the rhamnolipid biosurfactants (a rhamnose-containing glycolipid) produced by Pseudomonas strains have received greater attention due to their remarkable tensioactive and emulsifying properties.^8–11

An amphiphilic rhamnolipid molecule is composed of two moieties; one half is the hydrophilic sugar part composed of mono- or dirhamnose, and the second half is hydrophobic lipid moiety possessing one or two saturated or unsaturated fatty acid residues. The lipid moiety is attached to the sugar by O-glycosidic linkage whereas the two 3-hydoxy acyl groups are joined together by the formation of an ester bond. Notably, several types of rhamnolipid isoforms are produced by microorganisms and their structural diversity is determined by the presence of number of rhamnose and/or fatty acid moieties.¹² The chain length of the constituent fatty acids of rhamnolipids has been found to vary from C8 to C14.¹³ Besides, several single fatty acid-containing rhamnolipid biosurfactants were also identified.^14–16 In addition, productions of novel mono and dirhamnolipid methyl esters (Rha-C8-C8ME and Rha-Rha-C8-C8ME) and rhamnolipids with alternative fatty acid chains were also reported.^17,18

Most of the sedimentary basins that might contain oil have already been explored and it is becoming increasingly difficult to discover new oilfields.¹⁹ Furthermore, there has been an intense competition among the fossil fuel producing companies that has necessitated maximizing the recovery factor (RF) from their oilfields as well as maintaining an economic oil production rate. This may be achieved by recovery of left over oil from wells after primary and secondary recovery processes by specialized enhanced oil recovery (EOR) techniques.²⁰ The use of chemical surfactants for EOR is undesirable as it is hazardous, costly and may adversely affect the environment.²¹ The use of microbes or microbial biosurfactant to enhance oil recovery, also known as microbial enhanced oil recovery (MEOR), has proved to be a better alternative to the currently available chemical surfactants because biosurfactants are generally less toxic and eco-friendly.^22–25 However, application of biosurfactants in microbial enhanced oil recovery depends on their stability at extreme conditions of temperature, salinity and pH, or surface activities.²⁶ There are reports describing the effect on bioremediation of crude oil contaminated oil fields by exogenously seeded biosurfactant producing microbes which were indigenous to those oil fields.³ In this study, an attempt was made to structurally characterize a major biosurfactant produced by P. aeruginosa strain NBTU-01 isolated from a petroleum-oil contaminated soil sample. Our second objective was proteomic identification of proteins and enzymes associated with biosynthesis and regulation of rhamnolipid biosurfactant production. Further, with an aim to mitigate the problem associated with tertiary recovery of left out oil from petroleum oil fields, the potency of crude rhamnolipid biosurfactant in enhanced oil recovery was also explored.

Experimental section

Materials

Aliphatic hydrocarbons hexene, dodecane, hexadecane and octadecane, were purchased from Merck-Schuchardt, Germany. Ethyl acetate and hexane were purchased from Merck India Ltd., India. Nutrient and Luria-Bertani (LB) agar were purchased from Hi-Media Ltd. India. Crude petroleum–oil hydrocarbons contaminated soil samples were obtained from oil-fields of Oil and Natural Gas Commission (ONGC) of Assam, India.

Methods

Isolation and screening of biosurfactant producing bacterial strain. Petroleum-oil contaminated soil samples (1.0 g) were weighed and serially diluted from 10⁻¹ to 10⁻⁷ in 0.9% sterile NaCl solution. A screening technique was developed to find out indigenous bacterial strains that readily adapt to moderate-climatic conditions and can efficiently produce biosurfactant. Pure cultures of bacteria were screened for their ability to grow on modified M9 medium supplemented with 1% glycerol or hexadecane as the sole source of carbon. The composition of modified M9 was (g l⁻¹) 6 g Na₂HPO₄, 3 gm KH₂PO₄, 1.0 gm NH₄NO₃, 0.5 gm NaCl, 0.246 gm MgSO₄·7H₂O, 0.014 gm CaCl₂ and 50 μg CuSO₄·7H₂O, 10 μg H₃BO₃, 10 μg MnSO₄·5H₂O, 70 μg ZnSO₄·7H₂O, 10 μg MoO₃, 1.0 mg FeSO₄·7H₂O.²⁶ Single bacterial colony isolation from turbid cultures was performed by plating appropriately diluted culture samples onto lysogeny broth (LB) medium composed of 10.0 g Bactotryptone (Difco), 5.0 g of yeast extract (Difco), and 10.0 g of NaCl in 1000 ml of distilled water (pH adjusted to 7.0) and supplemented with 15.0 g of Bacto Agar (Difco).

The bacterial strains were sub-cultured on LB agar plates before used as inoculums and stored in glycerol at −80 °C. A total of 5 bacterial isolates, some of which having the ability to produce biosurfactant, NBTU-01, G#85, G#207, G#191 and W2 were isolated and pure culture was maintained. Pure cultures of bacteria were screened for their ability to grow on modified M9 medium supplemented with 1% (v/v) hexadecane or glycerol as the sole source of carbon at 37 °C for 24 h at 150 rpm in an orbital shaking incubator.² Bacterial growth in any of the above medium was determined by measuring the bacterial cell population at 600 nm, dry biomass,²⁷ extracellular protein content²⁸ as well as surface tension reducing property of cell-free culture supernatant.²⁷ Surface tension measurement assay was performed for screening of biosurfactant producing bacteria.² Briefly, cell-free culture supernatant (CFS) was obtained by centrifuging the fermentative broth at 10 000 rpm for 20 min at 4 °C (Multifuge 1XR centrifuge, Thermo Scientific, USA) and surface tension reducing property of CFS was measured at 25 °C using du Nouy ring tensiometer (JENCON, India). Surface tension reducing property of un-inoculated medium was considered as a control.²⁹ On the basis of the surface tension reduction property of CFS, the best strain was selected for further study.

Identification of bacterial strain. Bacterial identification was done by using the standard microbial identification procedure viz. (a) studying the morphological characteristics, biochemical tests and phenotypic properties of bacteria, and (b) 16S rRNA gene sequencing followed by its phylogenetic analysis.¹

For ribotyping, the bacterial genomic DNA was isolated and 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal primers that were designed to amplify a 1538-bp segment of the 16S rDNA.³⁰ The forward primer was: 5′-GAG TTT GAT CCT GGC TCA G-3′, and the reverse primer was: 5′-CGG CTA CCT TGT TAC GAC TT-3′. PCR was carried out as described by Rahman et al.,³¹ in PCR system thermal cycler (GeneAmp© PCR system 9700, Applied Biosystems). The purified PCR product (1.5 kb) was used directly for the automated DNA sequencing using 3130 Genetic Analyser (Applied Biosystem, Switzerland). The deduced sequence was subjected to BLAST search tool from the National Centre for Biotechnology Information, Bethesda, MD, USA (http://www.ncbi.nlm.nih.gov) to retrieve homologous sequences in GenBank.

The 16S rDNA sequence of bacteria under study was aligned with reference sequences showing sequence homology from the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the multiple sequence alignment programme of MEGA 6.³² Phylogenetic trees were constructed by distance matrix-based cluster algorithms viz. unweighted pair group method with averages (UPGMA), neighbour joining, maximum-likelihood and maximum-parsimony analyses.^33–35

Screening of fermentation conditions for efficient biosurfactant production. To study the effect of different carbon sources on biosurfactant production glucose, maltose, sucrose, glycerol, dodecane, or octadecane was supplemented to modified M9 medium at a concentration of 1% (either w/v or v/v). The following five inorganic and organic nitrogenous sources at a concentration of 0.1% (w/v) were tested for their effect on biosurfactant production by the bacterial isolate-peptone, ammonium chloride, potassium nitrate, tryptone and ammonium nitrate. The optimum pH for growth and biosurfactant production was determined by adjusting the pH of the modified M9 medium from 3.0 to 11.0.²⁹ The bacteria were grown at 37 °C, 150 rpm and the surface tension reduction of CFS as well as bacterial biomass were determined at an interval of 24 h.

Isolation and purification of rhamnolipid biosurfactant from P. aeruginosa strain NBTU-01. For biosurfactant production, 1 ml of the mid-logarithmic culture of bacteria (absorption at 600 nm ∼0.5) was inoculated into a 1L conical flask containing 500 ml sterile M9 medium supplemented with 1% (v/v) glycerol, 0.1% ammonium nitrate, pH 6.8 and incubated at 37 °C at 150 rpm on an orbital shaker (Scigenics Biotech, India) for 96 h. The crude rhamnolipid biosurfactant was isolated from CFS as described previously³⁶ and fractionated by Silica gel column chromatography using chloroform/methanol mobile phases as described by Deepika et al.³⁷ The fractions eluted by 50 : 50 (v/v) chloroform/methanol showed best surface tension reducing activity. These fractions were pooled and dried under vacuum in a rotor evaporator at 40 °C. This pooled fraction was referred to as partially purified rhamnolipid biosurfactant.

The partially purified rhamnolipid biosurfactant was then subjected to preparative thin-layer chromatography (TLC) and rhamnolipid biosurfactant spots were detected by exposure to iodine vapor.²⁴ The silica gel scraping of the major spot was collected and the rhamnolipid biosurfactant was extracted three times with 8 ml of CHCl₃/CH₃OH (1 : 2 v/v). The solvent scraping mixture was vortexed for 1 min, centrifuged for 10 min at 10 000 rpm and then the supernatant containing biosurfactant was carefully transferred to a sterile tube, dried under vacuum and stored at 4 °C for further analysis.

Structural characterization of rhamnolipid biosurfactant. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was employed to determine the molecular mass and elucidation of chemical structures of rhamnolipid molecules. The chemical structure of the TLC purified major biosurfactant was determined by Fourier Transform Infrared (FT-IR) spectroscopy and Nuclear Magnetic Resonance (NMR) study.

UV-Vis spectral and FTIR analysis. A UV-Visible spectrum of TLC purified biosurfactant (100 μg ml⁻¹ in 50 mM sodium carbonate) was recorded in the spectral range of 200–800 nm (Multiskan Go, Thermo scientific, USA). For FTIR analysis, the TLC purified biosurfactant sample (0.3–0.5 mg) was dispersed uniformly in spectral grade KBr. FTIR analysis was performed using Nicolet Impact I-410 FTIR spectrometer within spectral range of 4000–400 cm⁻¹ as described by Das and Mukherjee.²²

LC-MS/MS analysis to identify and quantitate the rhamnolipid biosurfactant. LC-MS/MS analysis was performed with an Agilent 6530 QTOF mass spectrometer using electrospray in negative ion mode. Briefly, 10 μg of partially purified rhamnolipid biosurfactant was dissolved in 10 μl of methanol : chloroform (50 : 50, v/v) and introduced into the mass spectrometer by an Agilent Technologies nanoHPLC equipped with an Eclipse Plus C18 (100 × 2.1 mm, 1.8 μm) reversed-phase HPLC column. An acetonitrile–water gradient containing 5 mM ammonium acetate was used, starting with 0–10% acetonitrile for 10 min, 85% acetonitrile from 10–34 min, 10% acetonitrile from 34–40 min and then finally hold for 5 min. The column temperature was maintained at 35 °C. The nanoHPLC flow rate was maintained 0.3 ml min⁻¹ and directly introduced into the mass spectrometer. Capillary and cone voltage was set at 3.5 kV and 500 V, respectively. The source and desolvating gas temperature was set at 150 and 300 °C, respectively. The scanning mass range was from 100 to 1700 Da. Collision-induced dissociation was performed with 35 psig of nitrogen in the collision cell, with collision energy of 22, 26 and 30 eV. Generated data was searched for the identity of compound in Agilent Mass Hunter METLIN AM PCD Database/PCDL library. For confident results, PCDL software scores the database matches based on the similarity of the mono isotopic masses (Mass Match), isotope ratios (Abundance Match), isotope spacing (Spacing Match), and optionally, the retention time (RT Match).

Quantification was performed by integration of the pseudomolecular ion of each compound as described by Lotfabad et al.²⁴ with slight modifications. Relative abundance (%) of each rhamnolipid congener was calculated as follows:

where, TIC-MS is the total ion chromatogram-mass spectroscopy.

The absolute quantification of a rhamnolipid congener per liter was calculated as:

where, 10 μg of partially purified rhamnolipid fraction was injected in LC/MS-MS system and 270.8 mg l⁻¹ was the total yield of the partially purified rhamnolipid fraction (see below).

¹H and ¹³C NMR analysis of purified biosurfactant. The TLC purified biosurfactant was characterized by ¹H and ¹³C NMR spectroscopic analysis (JEOL, ECS-400 MHz NMR, version 4.3.6) CDCl₃ as the solvent with TMS as reference.²³ The biosurfactant was dissolved in CDCL₃ (50 mg ml⁻¹) and the spectra were recorded at 70 °C with 5000–5200 accumulations, 5.9 μs pulse duration, 1.2 s acquisition time and 6 μs relaxation delay.³⁸

Differential scanning calorimetric (DSC) analysis of purified biosurfactant. DSC analysis of purified biosurfactant was done with thermal analyzer system (DSC-60, Shimadzu). Eight mg of dried sample was loaded on a platinum pan and the DSC analysis was done by gradually increasing the temperature from 10 to 300 °C with a temperature gradient of 10 °C min⁻¹ under a nitrogen atmosphere and heat flow against each point of temperature was recorded.^38,39

Proteomic analysis of bacterial intracellular proteins. The pure culture of P. aeruginosa strain NBTU-01 was inoculated in 100 ml of M9 medium containing 1% (v/v) glycerol and incubated at 37 °C on a rotary shaker at 150 rpm. After 96 h, bacterial culture was centrifuged at 6000 rpm for 10 min at 4 °C (Heraeus Multifuge X1R Centrifuge, Thermo Scientific, USA). The pellet was washed three times with 20 mM Tris–HCl buffer, pH 8.0 and thereafter bacterial cells were re-suspended in 0.5 ml of above buffer containing cocktail of protease inhibitors (Sigma-Aldrich) to prevent the proteolysis during subsequent steps of protein extraction. The cells were then subjected to four cycles of freeze–thaw followed by 6 cycles of sonication in an ice-bath for 30 s with an interval of 1 min in between. The cell lysate was centrifuged at 12 000 rpm for 30 min at 4 °C and the supernatant containing bacterial intracellular proteins was passed through 0.2 μm filter, the filtrate was desalted and the protein concentration of the filtrate was determined.²⁸

Bacterial intracellular proteins were identified by LC-MS/MS analysis.^40,41 Briefly, 40 μg of bacterial intracellular proteins were reduced with 10 mM dithiothreitol (DTT) for 15 min at 56 °C followed by alkylation with 55 mM iodoacetamide (IAA) for 1 h at room temperature in the dark. The reduced and alkylated proteins were subjected to in-solution digestion with proteomics grade trypsin (50 ng μl⁻¹ in 25 mM ammonium bicarbonate containing 10% acetonitrile) for 18 h at 37 °C. The digested peptides were dried, reconstituted in 15 μl of the 0.1% (v/v) formic acid and then subjected to RP-nano HPLC (Agilent 1200 HPLC)-MS/MS (Thermo LTQ-Orbitrap Discovery) analyses. The ion source was ESI (nano-spray), fragmentation modes were collision induced dissociation (y and b ions), MS scan mode was FT-ICR/Orbitrap, while MS/MS scan mode was linear ion trap. For fixed and variable modifications, carbamidomethylation of cysteine residues and oxidation of methionine residues, respectively, were selected. The MS/MS data were searched in National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) database against P. aeruginosa proteins using PEAKS 7.0 software. The identified proteins were classified and grouped according to their biological function(s). To avoid multiple comparison artifacts generated through shotgun proteomics approach and to eliminate erroneous identification of P. aeruginosa proteins, the false discovery rate (FDR) was kept very stringent (0.8%). Further, only matching peptides and proteins showing a −10 lg P value ≥30 and 20, respectively, were considered for identification purposes. To identify the occurrence of putative conserved domain(s), if any, in the identified regulatory proteins and enzymes responsible for rhamnolipid biosynthesis, the MS-MS derived peptide sequences were searched in NCBI databases using BLASTp programme (https://www.ncbi.nlm.nih.gov).

Biochemical and physiochemical characterization of rhamnolipid biosurfactant.
Surface tension and CMC measurement. Critical micelle concentration (CMC) and reduction in surface tension were measured by the Du Nouy ring method in a surface tensiometer (JENCON, India).⁴² The surface tension measurement was carried out at room temperature (∼23 °C) after dipping the platinum ring in deionized water for a while in order to attain equilibrium conditions. The instrument was calibrated against un-inoculated M9 medium and 50 mM sodium carbonate, separately for the surface tension measurement of crude (CFS) and purified biosurfactant, respectively. All the measurements were performed in triplicate at room temperature (∼23 °C). The CMC value was then determined from the break point of the surface tension versus its log of bulk concentration curve.¹
Emulsification index of biosurfactant. Emulsification assay of partially purified biosurfactant was carried out according to Cooper and Goldenberg and as modified by Mukherjee.^29,43 Briefly, the emulsification activity was measured by adding 6.0 ml of oil (vegetable oil/crude petroleum oil/kerosene/diesel) to 4.0 ml of biosurfactant sample (100 μg ml⁻¹ in 50 mM Na₂CO₃) and vortexing the mixture at a high speed for 2 min. The resulting mixture was kept at room temperature for 24 h and the emulsification index (E₂₄) was calculated as follows:

Stability studies. Crude or purified rhamnolipid biosurfactant was dissolved in 0.5 M sodium carbonate solution at its CMC value and it was heated to 100 °C for different time intervals (5 to 300 min), cooled to room temperature followed by measuring the surface tension activity of biosurfactant solution. The surface tension activity of unheated biosurfactant solution was considered as 100% (control) and other values were compared to that. The pH stability was studies by adjusting the pH (3.0 to 12.0) of biosurfactant solutions and the surface tension of biosurfactant at different pH was measured. The pH at which the biosurfactant displayed highest surface tension reducing property was considered as 100% and other values were compared with that.²⁹

Removal of crude petroleum oil from sand pack column. The potential application of the biosurfactant in enhanced-oil recovery (EOR) was evaluated using the ‘sand pack column’ technique as described by Abu-Ruwaida et al.,⁴⁴ and as modified by Das and Mukherjee.¹ A glass column (61 cm × 3.5 cm) was packed with 150 g of acid washed dry sand. The brine solution (5% NaCl, w/v) was then passed through the column and pore volume (PV) was determined by measuring the volume required to make the sand matrix wet in brine solution. To ensure 100% saturation three PVs of brine solution was passed through the column. After saturating column with brine solution, 50 ml of crude petroleum oil (obtained from ONGC, Assam) was passed through the column under pressure until the column got saturated with oil. The potential of the crude rhamnolipid biosurfactant for enhanced oil recovery was evaluated at room temperature (∼23 °C) by pouring 50 ml crude rhamnolipid biosurfactant solution (100 μg ml⁻¹ in 50 mM sodium carbonate) in the column at a constant flow rate of 0.6 ml min⁻¹ by using a peristaltic pump. Recovery of petroleum-oil from sand pack column by washing with 50 mM sodium carbonate under identical experimental conditions served as a control. The actual volume of oil released by crude rhamnolipid biosurfactant was determined by subtracting the volume of oil released by sodium carbonate.

In an another set of experiment, crude rhamnolipid biosurfactant was heated at 100 °C for 5 h before assessing its oil releasing potency from sand pack column. The volume of oil recovered from sand pack column by unheated crude biosurfactant served as a control. The percent oil recovery from column was evaluated by the following equation.⁴⁵

Oil recovery (%) = (M₂/M₁) × 100

where M₁ is oil content in the sand (ml), and M₂ represents oil eluted (ml) by crude rhamnolipid biosurfactant.

Statistical analysis. The experimental data are presented in terms of arithmetic mean ± standard deviations of three replicates. The significance of difference between two values was determined by Student's “t” test. Statistical analysis was done using Sigma Plot software, version 10.0 (SPSS Inc., UK).

Results and discussions

Isolation and characterization of a biosurfactant producing bacterium

In this study, a total of 5 biosurfactant producing potential bacterial isolates were isolated and their pure cultures were maintained. Growth and surface tension reducing property of CFS of the bacterial isolates when grown in hexadecane-supplemented or glycerol-supplemented culture medium are presented in ESI Table S1.† It was observed that the bacterial isolate NBTU-01 exhibited maximum growth and surface tension reduction of culture medium from 68.0 mN m⁻¹ to 31.5 mN m⁻¹ after 96 h of growth in glycerol supplemented modified M9 medium (ESI Table S1†). Therefore, isolate NBTU-01 was considered as promising bacterium for efficient biosurfactant production and pure culture of this isolate was used for subsequent studies.

The phenotypic characterization of strain NBTU-01 is shown in ESI Table S2.† The biochemical and morphological characteristics of the isolated strain revealed that it might belong to the genus Pseudomonas. The 16S rDNA sequence (accession no. JQ241432) of the strain NBTU-01 showed 99% sequence similarity with its closest phylogenic neighbor P. aeruginosa (accession no. KU870749) (ESI Fig. S1†). Therefore, this bacterium was identified as strain P. aeruginosa strain NBTU-01.

Effect of fermentation conditions on biosurfactant production

Glycerol and ammonium nitrate served as the best carbon and nitrogen source, respectively for optimum biosurfactant production by P. aeruginosa strain NBTU-01 (data not shown). The maximum reduction in surface tension was observed when the bacteria were grown at pH 6.8. Therefore, these conditions were chosen for biosurfactant production. However, kinetics of biosurfactant production suggested that optimum reduction in surface tension was achieved 96 h post incubation and thereafter, no further significant increase in biosurfactant production was detected. The above observations are in close agreement with our previous report on optimum conditions for biosurfactant production by P. aeruginosa mucoid and non-mucoid strains.¹

Purification of rhamnolipid biosurfactant

About 3.14 g of crude rhamnolipid biosurfactant was isolated by acid precipitation of 1L CFS of P. aeruginosa strain NBTU-01 and this yield was comparable to biosurfactant production by other Pseudomonas species.^23,46–48 The surface tension reducing property of CFS, crude biosurfactant, and biosurfactant fractions eluted with different ratios of CHCl₃/CH₃OH (v/v) from Silica gel G-60 is shown in Table 1. The fraction eluted with 50 : 50 (v/v) CHCl₃/CH₃OH showed maximum surface tension reducing property and this fraction was termed as partially purified rhamnolipid biosurfactant [Table 1]. The yield of this fraction was determined at 270.8 mg l⁻¹ which represented 8.62% yield [Table 1]. TLC separation of the above fraction showed a major dark yellow spot of dirhamnolipid^24,35 (R_f value of 0.30) and a minor faint spot of monorhamnolipid (R_f value 0.49) [ESI Fig. S2†]. The yield of dirhamnolipid was found to be 212.2 mg l⁻¹ which was 6.75% yield of crude biosurfactant [Table 1]. The surface tension reduction value of purified dirhamnolipid was found to be 29.5 ± 0.7 nM m⁻¹ [Table 1] and it was selected for further studies.

Table 1 Summary of isolation and purification of a major biosurfactant from P. aeruginosa strain NBTU-01. Values are mean ± SD of triplicate determinations^a

Fractions	Surface tension (mN m^−1)	Dry amount recovered (mg l^−1)	Yield%	CMC (mg l^−1)
a ND: not determined.
Cell free supernatant	31.5 ± 0.29	ND	—	ND
Crude biosurfactant	31.5 ± 1.0	3140.0 ± 6.24	100	110 ​± 2.0
Silica gel chromatography (chloroform: methanol mobile phase)
50 : 3	35.9 ± 0.6	920.34 ± 1.73	29.31	ND
50 : 5	36.2 ± 1.2	370.60 ​± 5.85	11.80	ND
50 : 50	29.8 ± 0.22	270.75 ​± 4.50	8.62	75 ​± 2.8
Thin layer chromatography
RL 1	29.5 ± 0.7	212.2 ± 4.43	6.75	72 ± 2.25

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Structural characterization

In general, rhamnolipid from P. aeruginosa strains have been detected in the mass range from m/z 331 to 677.^1,2,49 The partially purified rhamnolipid biosurfactant was subjected to LC-MS/MS analysis. Mass spectrometry analysis based on MFG scoring for different isotopes and abundances revealed the presence of 8 rhamnolipid congeners in partially purified biosurfactant. The relative abundance (%) of each rhamnolipid congener in the mixture was obtained from the relative area of their corresponding pseudomolecular ions,²⁴ and their relative abundance was found to vary from 42% to 1.3% [Table 2]. The MS/MS profiles of major analogues detected at m/z 649.37 (41.55%) [Fig. 1(a)] and 677.40 (25.33%) [Fig. 1(b)] correspond to the deprotonated molecules of Rha-Rha-C10-C10 and Rha-Rha-C12-C10, respectively. From the LC-MS/MS analysis, the yield of the Rha-Rha-C10-C10 and Rha-Rha-C12-C10 was determined to be 112.18 mg l⁻¹ and 68.39 mg l⁻¹, respectively [Table 2]. The LC-MS/MS analysis confirms the predominance of dirhamnolipid (78.41%) over monorhamnolipid (21.59%) in the TLC purified biosurfactant [Table 2].

Table 2 LC-MS/MS analysis to determine the structure, relative abundance, and quantity of rhamnolipid homologues produced by P. aeruginosa strain NBTU-01

Pseudomolecular ion (m/z)	Mass	Congener	Type of rhamnolipid	Formula	Relative abundance (%)	Quantity (mg l^−1)
649.37	650.38	Rha-Rha-C10-C10	Dirhamnolipid	C32H58O13	41.56	112.18
677.44	678.41	Rha-Rha-C12-C10	Dirhamnolipid	C34H62O13	25.33	68.39
503.32	504.32	Rha-C12-C8	Monorhamnolipid	C26H48O9	11.04	30.78
655.43	656.44	Decenoyl-Rha-C10-C10	Dirhamnolipid	C36H64O10	9.43	25.46
531.35	532.35	Rha-C12-C10	Monorhamnolipid	C28H52O9	5.67	15.30
529.33	530.34	Rha-C12:1-C10	Monorhamnolipid	C28H50O9	3.63	9.80
705.44	706.45	Rha-Rha-C12-C12	Dirhamnolipid	C36H66O13	2.09	5.63
385.22	386.23	Rha-C14:2	Monorhamnolipid	C20H34O7	1.27	3.42

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Fig. 1 LC-MS/MS characterization of partially purified biosurfactants (a) mass spectra of the fragmented pseudomolecular ion at m/z 649.39 of congener Rha-Rha-C10-C10 and the daughter ions generated by fragmentation. (b) Mass spectra of the fragmented m/z 677.44 pseudomolecular ions of congener Rha-Rha-C12-C10 and the daughter ions generated by fragmentation.

In order to determine the structure and the position of the fatty acids, the rhamnolipid components were analyzed by the tandem-MS mode. The main ion of first major component (m/z 649.37, Rha-Rha-C10-C10) showed major daughter ions at m/z 169.12 and 479.24 [Fig. 1(a)]. This indicates rupture of the ester bond between the alkyl chains, releasing Rha2-C10 (m/z 479) and hydroxyl decanoate (m/z 169). A daughter ion at m/z 143, corresponding to a single rhamnose molecule was also identified [Fig. 1(a)]. The MS/MS analysis of the other major component of rhamnolipid biosurfactant (m/z 677.44, Rha-Rha-C12-C10) apart from showing two daughter ions at m/z 479.24 and 169.12, also demonstrated a signal at m/z 507.27 which corresponds to Rha2-C12 [Fig. 1(b)]. It has been suggested that for rhamnolipid congeners containing two fatty acid moieties of different chain lengths or containing double bonds, the congener containing shorter chain or shorter saturated chain adjacent to the sugar moiety will be the most abundant congener.⁵⁰ Majority of the biosurfactants produced by P. aeruginosa strains with different carbon sources were reported to be dirhamnolipid^48,51 and only a few reports showed that monorhamnolipid was produced as predominant biosurfactant by Pseudomonas sp.⁵² The chemical composition of the rhamnolipid mixtures produced on different carbon sources (2% glycerol, glucose, n-hexadecane, or n-alkanes) did not vary.⁵³ As shown in ESI Table S3,† the predominance production of the rhamnolipid Rha-Rha-C10-C10 on glycerol medium correlates well with earlier reports.^53–55

UV-Visible spectra of the TLC purified major biosurfactant of P. aeruginosa strain NBTU-01 showed absorption maxima at 230 and 270 nm which can be attributed to the π–π* and n–π* transition of carbonyl group ([double bond splayed left]C=O), respectively (ESI Fig. S3†). FTIR analysis of the purified rhamnolipid biosurfactant demonstrated a broad absorption peak at 3365 cm⁻¹ which was the characteristic stretching or vibration mode of –OH group [Fig. 2(a)]. The sharp absorption peak at 1732 cm⁻¹ was due to the C=O stretching of the ester linkage, whereas the peak at 1671 cm⁻¹ was due to the C=O stretching frequency of the carboxylate anion.⁵³ The peaks at 1457–1336 cm⁻¹ were due to the –C–H bending that indicated the presence of aliphatic chain in the rhamnolipid structure. The absorption peaks at 1319–1052 cm⁻¹ were due to the stretching vibration of C–O–C and C–OH linkages that confirmed the formation of bond between carbon atom and hydroxyl group in the rhamnose ring of rhamnolipid biosurfactant [Fig. 2(a)]. The C–H symmetric and asymmetric stretching vibration of –CH₂ group of the aliphatic chain appeared at 2926 cm⁻¹ and 2857 cm⁻¹, respectively.⁷ The FTIR spectral characteristics are consistent with the published reports.^56,57

Fig. 2 Determination of structure of TLC purified major rhamnolipid biosurfactant produced by P. aeruginosa strain NBTU-01 by (a) FTIR spectra. The characteristic absorbance bands at 3365 cm⁻¹ (–OH group stretching due to hydrogen bonding), 1732 cm⁻¹ (C=O stretching of the ester linkage), 1671 cm⁻¹ (C=O stretching carboxylate anion), 1457–1336 cm⁻¹ (aliphatic chain), 1319–1052 cm⁻¹ (C–O–C and C–OH stretching in the rhamnose), 2926 cm⁻¹ and 2857 cm⁻¹ (the aliphatic bonds –CH₃, –CH₂, and –C–H stretching) of purified rhamnolipid. (b) ¹H NMR spectra. The chemical shift at 0.88, 1.33, 2.41, 4.20, 4.89, 5.40 and 8.19 ppm corresponds to characteristics peaks of L-rhamnose moiety and aliphatic moiety of purified rhamnolipid. (c) ¹³C NMR spectra. The chemical shift at 14.15, 22.67, 30.98, 31.82, 77.12 and 207.14 ppm corresponds to different carbon atom of pyranose ring, aliphatic chain, carbonyl and carboxylic groups of purified rhamnolipid. (d) Predicted structure of purified dirhamnolipid molecule.

The detail chemical structure of the TLC purified biosurfactant was determined by NMR study. ¹H NMR spectrum of the purified biosurfactant is presented in Fig. 2(b). The protons of the two main compositions (L-rhamnose moiety and aliphatic moiety) of the biosurfactant were detected. The peaks at 0.88 ppm and 1.33 ppm were due to the proton of –CH₃ and –CH₂ moiety of aliphatic chains, respectively. Peak at 2.41 ppm corresponds to the methylene (–CH₂) proton attached to the [double bond splayed left]C=O group. A typical signal of oxymethine (O–CH) proton was observed at 4.20 ppm and 4.89 ppm. The chemical shift (δ) value for the methine proton of O–CH–O linkage was observed at 5.40 ppm. In addition, a peak at 8.19 ppm was observed due to the proton of carboxylic (–COOH) group of the biosurfactant [Fig. 2(b)].

Furthermore, the structural characteristic of the purified rhamnolipid was studied by ¹³C NMR analysis and the spectrum is shown in Fig. 2(c). The peak at 14.15 ppm and 22.67 ppm represents the carbon atom of methyl moiety of the pyranose ring and aliphatic chain, respectively. Moreover, peaks at 30.98 ppm and 31.82 ppm correspond to the carbon atom of methylene (–CH₂) moiety bonded to the [double bond splayed left]C=O group. The carbon atoms (bonded to –OH groups) of pyranose ring were identified by the peak at 77.12 ppm. The peak at 207.14 ppm due to the carbonyl carbon confirmed the presence of the –COOH group [Fig. 2(c)].

Based on the NMR study, we proposed a hypothetical structural elucidation which implies that the purified biosurfactant consists of a major proportion of fatty acids followed by repeating units of rhamnose sugar. These findings are consistent with the structure of the purified biosurfactant as dirhamnolipid and its derived proposed structure is L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate [Fig. 2(d)].

DSC analysis of rhamnolipid

The DSC thermogram showed that crystallization temperature (T_c) of purified rhamnolipid was determined at ∼99 °C and its melting point peak was observed at 134 °C [Fig. 3]. These results indicated that the transition of rhamnolipid biosurfactant from an amorphous solid to a crystalline solid was an exothermic process. No characteristics degradation peak of rhamnolipid was observed after heating it at 300 °C which advocated the industrial application of this biosurfactant in high temperature oil well.

Fig. 3 DSC thermogram of TLC purified dirhamnolipid biosurfactant obtained from P. aeruginosa strain NBTU-01.

Proteomic analysis to identify intracellular proteins involved in biosynthesis and regulation of rhamnolipids production

Rhamnolipid biosynthesis is a complex biological phenomena and it involves the sequential action of several biosynthetic enzymes and regulation factors.⁵⁸ Although culture conditions influence the biosynthesis of a large diversity of RL congeners and homologs albeit in liquid culture, two major types of rhamnolipid molecules (1) monorhamnolipid, rhamnosyl-b-hydroxydecanoyl-b-hydroxydecanoate (Rha-C10-C10), and (2) dirhamnolipid, rhamnosyl-rhamnosyl-b-hydroxydecanoyl-b-hydroxydecanoate (Rha-Rha-C10-C10) were reported to be produced by P. aeruginosa.⁵⁰ The biosynthesis of rhamnolipids occurs in three different steps-(i) biosynthesis of lipid moiety, (ii) biosynthesis of rhamnose and other sugars, and (iii) dimerization and sequential rhamnosylation reaction to produce rhamnolipid molecules.⁵⁸ The complex process of rhamnolipid biosynthesis is attributed to three key enzymes RhlA, RhlB and RhlC.^59–62 RhlA, a 32.5 kDa protein of P. aeruginosa localized in the periplasm catalyzes the synthesis of the fatty acid dimer moiety of rhamnolipids and free 3-(3′-hydroxyalkanoyloxy) alkanoic acids (HAAs).^59,61 The RhlB (MW 47 kDa) is a rhamnosyltransferase enzyme catalyzes the formation of mono-rhamnolipids using dTDP-L-rhamnose and an HAA as a precursor molecule.⁶⁰ The third protein RhlC (35.9 kDa) is an inner membrane bound enzyme specific for dirhamnolipid production and its major role is biosynthesis of lipopolysaccharide (LPS).^60,62 Another enzyme, β-ketoacyl reductase (RhlG)⁶³ is presumed to be involved in rhamnolipid biosynthesis,⁶⁰ but Zhu and Rock showed that this enzyme did not have any role in rhamnolipid production and its physiological substrate is yet to be identified.⁶⁴

By LC-MS/MS analysis key enzymes required for L-rhamnose production⁶² viz. glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase (RmlC), and dTDP-4-keto-6-deoxy-L-mannose reductase (RmlD) as well as for rhamnolipid biosynthesis (RhlB and RhlC) were identified in P. aeruginosa strain NBTU-01 [ESI Table S4†]. In addition, several other transcription factors whose function is to regulate the rhamnolipid biosynthesis in P. aeruginosa strain NBTU-01 were also identified [ESI Table S4†]. The alignments of MS-MS derived peptide sequences with rhamnolipid biosynthesis enzymes are shown in ESI Table S5.† Interestingly, proteomics analysis did not ascertain presence of RhlA in the bacterium under study which may presumably due to labile nature of this enzyme.⁵⁹ The MS/MS peptide sequence GIILAGGSGTR (m/z 501.292) of P. aeruginosa strain NBTU-01 showing homology with RmlA protein demonstrated putative conserved domain of Glyco_tranf_GTA_type superfamily [ESI Table S5†]. This superfamily includes the glycosyltransferases (GTs) that synthesize oligosaccharides, polysaccharides, and glycoconjugates by transporting sugar moiety from activated nucleotide-sugar to a growing chain of oligosaccharide, a lipid, or a protein.

The regulation of rhamnolipid biosynthesis is also a complex phenomenon. Studies have shown the impact of various direct and indirect biotic and/or abiotic factors like quorum sensing, nutritional status or stress response on rhamnolipid biosynthesis by P. aeruginosa.⁵⁸ It has also been demonstrated that vast number of transcriptional factors also regulate the rhamnolipids production.^58,65,66 The proteomic analysis has provided an overview of synthesis of various intracellular proteins and enzymes during the growth of bacterium on glycerol. Several of these proteins and enzymes play a significant role in RL biosynthesis. By LC-MS/MS analysis a total of 206 intracellular proteins including the enzymes responsible for rhamnolipid biosynthesis were identified in P. aeruginosa strain NBTU-01. These intracellular proteins and/or enzymes are associated with metabolism of carbohydrate (glycolysis, gluconeogenesis, and citric acid cycle), regulation of transcription, and translation (protein biosynthesis), protein folding, transport of metabolites, stress response, biosynthesis of nucleotide, fatty acids, amino acids, and porphyrins, cell division, cell adhesion, DNA condensation, electron transport, and biological oxidation–reduction [ESI Table S4†]. Synthesis of several transcriptional (AlgR) and post-transcriptional (GidA and DskA) factors which belong to LytR/AlgR, MnmG and DksA protein families, respectively and play an important role in regulation of rhamnolipid biosynthesis was also revealed by proteomics analysis of P. aeruginosa strain NBTU-01 [ESI Table S4†]. AlgR regulates the function of AlgC which is associated with the production of an enzyme involved in lipopolysaccharide (LPS) synthesis (phosphoglucomutase activity)⁶⁷ and alginate production (phosphomannomutase activity).⁶⁸ AlgC may also play a crucial role in the production of rhamnolipid.⁶⁹ In the absence of AlgR, expression of AlgC was found to reduce approximately five folds.⁶⁸

GidA protein controls rhl-dependent quorum sensing (QS) gene expression by modulating RhlR expression post-transcriptionally.⁷⁰ This protein is involved in tRNA modification and shares homology with conserved flavin adenine dinucleotide binding proteins.^71–73 Another transcriptional regulator, DksA is an exponential growth phase regulator and contributes to rhlAB translation at the basal level during stationary phase.^62,73 By proteomics analysis both GidA and DksA were identified in P. aeruginosa strain NBTU-01 [ESI Table S4†].

Based on proteomics analysis a schematic presentation of rhamnolipid biosynthesis by P. aeruginosa strain NBTU-01 has been proposed [Fig. 4]. Glycerol supplied in the medium feeds into the central carbon metabolism at the glyceraldehyde-3-phosphate level and thus pentose phosphate (PP) pathway is not utilized which ultimately wastes carbon via CO₂ production.⁷⁴

Fig. 4 Schematic presentation of rhamnolipid biosynthesis by the P. aeruginosa strain NBTU-01.

CMC and surface tension reduction

Rhamnolipid molecules show a wide range of CMC values, from 230 mg l⁻¹ for a mixture of seven rhamnolipid isomers⁷⁵ to 30 mg l⁻¹ for the rhamnolipids synthesized by P. aeruginosa L2-1 from cassava wastewater.⁵ The CMC value of crude and TLC purified rhamnolipid biosurfactant from the P. aeruginosa strain NBTU-01 was determined at 110 ± 2.0 and 72 ± 2.25 mg l⁻¹, respectively [Table 1]. At this CMC value, purified biosurfactant reduced the surface tension of water from 72.2 mN m⁻¹ to 29.5 mN m⁻¹ [Table 1]. Therefore, the dirhamnolipid biosurfactant under study exhibited excellent surface activity at a low CMC and this value was significantly lower as compared to the same property exhibited by rhamnolipid biosurfactant produced by P. aeruginosa⁷⁶ and biosurfactant obtained from Acinetobacter baylyi.²⁵ Notably, the most prevalent chemosynthetic surfactant sodium dodecyl sulfate (SDS) also shows higher CMC value at 2100 mg l⁻¹.⁷⁷

The emulsion formation property of purified dirhamnolipid biosurfactant is displayed in Table 3. Results showed that purified rhamnolipid biosurfactant was a good emulsifier of coconut oil and crude petroleum-oil with an E₂₄ value of 54.76 and 51.94, respectively [Table 3]. Higher E₂₄ value indicates excellent emulsifying capacity of the biosurfactant. The E₂₄ value of dirhamnolipid in this study is in good agreement with the E₂₄ values of biosurfactants produced by other Pseudomonas species.^23,78–80 The rhamnolipid under study effectively emulsified and stabilized the emulsions with vegetable oils and this property may be useful for making oil/water emulsions for food and improves the texture and creaminess in dairy products such as soft cheese and ice creams.⁸¹ Furthermore, appreciable emulsification property with petroleum-oil also suggested the potential use of P. aeruginosa strain NBTU-01 rhamnolipid biosurfactant for enhanced oil recovery.⁸²

Table 3 Emulsification index (E₂₄) of partially purified rhamnolipid biosurfactant from P. aeruginosa strain NBTU-01. Values are mean ± SD of triplicate determinations

Test oil	Emulsification index (E24)
Coconut oil	54.76 ± 1.7
Petroleum oil	51.94 ± 1.5
Diesel oil	44.87 ± 1.1
Kerosene oil	33.75 ± 1.8
Olive oil	4.82 ± 2.1
Mustard oil	2.44 ± 2.0

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Stability studies

The effect of pH on surface activity of different microbial biosurfactants has been well studied. The crude rhamnolipid biosurfactant from P. aeruginosa strain NBTU-01 was found to be reasonably stable over a pH range from 6.0 to 9.0; however, its surface tension reduction activity was significantly higher at pH 6.0 as compared to pH 9.0 [ESI Table S6†]. Conversely, at lower pH (<6.0) or at higher alkaline pH (>9.0) the rhamnolipid biosurfactant lost its surface active property indicating extreme alkaline or acidic conditions has a detrimental effect on its surface tension reducing property [ESI Table S6†]. The instability of P. aeruginosa biosurfactant below pH 6.0 has already been reported and it may be correlated to the presence of negative charged groups at the polar ends of rhamnolipid biosurfactant molecules, which are protonated under acidic condition.⁸³

Environmental factor such as pH and temperature play a crucial role in influencing the effectiveness of rhamnolipids.⁸⁴ Ilori et al.,⁸⁵ demonstrated that the chemical structure of biosurfactants provided an advantage to the degradation of hydrocarbons, but it can probably be destroyed under extreme conditions of temperature and pH. The crude rhamnolipid biosurfactant in our study remained stable without any change in its surface tension reduction property post incubation at 100 °C for 5 h [ESI Table S7†]. According to Desai and Banat, heat treatment (autoclaving at 121 °C for 15 min) had no appreciable effect on the emulsifying activities of some biosurfactants.¹⁸ Noteworthy to mention, several significant practical, economic and technical challenges need to be addressed before MEOR technologies can be deployed in the oil field. Biosurfactant flooding was ruled out because of the high reservoir temperature (97 to 115 °C).¹⁹ However, our study suggests that dirhamnolipid biosurfactant can thrive in very extreme conditions in oil reservoirs and this property is very advantageous for its applications in tertiary oil recovery and in the bioremediation of petroleum-oil polluted environment.³

Recovery of crude petroleum-oil from the sand packed column

Application of biosurfactants in microbial enhanced oil recovery depends on their stability at extreme conditions of temperature, salinity and pH, or surface activities.²⁶ Stimulation of microorganisms that produce biosurfactants and degrade heavy oil fractions in situ reduces the capillary forces that retain the oil into the reservoir and decreases oil viscosity, thus promoting oil flow thus resulting in increase in quantum of oil production.⁸³ Therefore, one of the targets of the present study was to evaluate the oil recovery property and stability of rhamnolipid biosurfactant produced by P. aeruginosa strain NBTU-01.

For obtaining a cost-effective process, the oil recovery experiment was done with crude rhamnolipid biosurfactant rather than TLC purified biosurfactant. The percentage of oil recovered by crude rhamnolipid biosurfactant from saturated sand pack column was found to be 70 ± 3.5% [Table 4]. In a sharp contrast, 50 mM sodium carbonate could elute only 10% of petroleum-oil from sand pack column under identical experimental conditions [Table 4]. This recovery of oil from saturated sand pack column by crude rhamnolipid biosurfactant in this study is higher than that shown by biosurfactant from B. subtilis⁸⁶ and P. aeruginosa strains³⁴ suggesting potential application of rhamnolipid biosurfactant produced by P. aeruginosa strain NBTU-01 in enhanced oil recovery.

Table 4 Percent petroleum-oil recovery from saturated sand pack column by crude rhamnolipid biosurfactant isolated from P. aeruginosa strain NBTU-01. Values are mean ± SD of triplicate determinations^b

Conditions	Oil released (%)
a Heated at 100 °C for 5 h.b Significantly higher with respect to control (*p< 0.001).
Control (water)	10 ± 1.9
Crude rhamnolipid biosurfactant	70 ± 3.5*
Crude rhamnolipid biosurfactant^a	64 ± 4.1*

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Conclusion

In this study, the partially purified rhamnolipid biosurfactant from P. aeruginosa strain NBTU-01 was chemically characterized as a mixture of 8 rhamnolipid congers. Among them, the most abundant rhamnolipid was identified as dirhamnolipid (Rha-Rha-C10-C10). The gene regulation of rhamnolipid production comprises a complex and highly controlled processes involving transcriptional and posttranscriptional regulations. The proteomics analysis of the isolate under study has provided an overview of the expression of different regulatory proteins and enzymes involved in rhamnolipid biosynthesis. Better understanding of the gene regulation and the environmental impact on expression of enzymes in rhamnolipid biosynthesis may contribute to obtain high yields of its industrial production. The rhamnolipid under study provided excellent properties in terms of thermostability, reduction of surface tension, low CMC value and exhibited an effective performance in oil recovery assays. Therefore, these findings suggested that the biosurfactant from the P. aeruginosa strain NBTU-01 could be a potential candidate to be used in the bioremediation of petroleum-oil contaminated sites and in situ oil recovery from oil wells (MEOR). This initial work paves the way to further exploit the potential of this strain across a broad range of industrial applications.

Acknowledgements

The authors thank Dr Ashish Pargaonkar, Agilent Technologies India Pvt. Ltd., Bangalore, and C-CAMP, NCBS, Bangalore for LC-MS/MS analysis of rhamnolipid and bacterial proteome, respectively, and Mr Anupam Saikia, Technical Assistant, ONGC-CPBT for some technical assistance. DG received fellowship from UGC-SAP (DRS-I) and TU, and PB was ONGC-CPBT project JRF. This project received partial financial support from ONGC-CPBT project “Proteomics study of aromatic hydrocarbons degradation enzymes of some selected bacterial strains prospecting strategies for environmental bioremediation” and UGC-SAP (DRS-I) to AKM.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra11979d

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