An Indigenous Biosurfactant Producing Burkholderia cepacia with High Emulsification Potential towards Crude Oil

In this study, the isolation of a potent indigenous biosurfactant-producing bacterium with potential value in enhanced bioremediation of oil-contaminated soil and oil tertiary recovery from the soil was investigated. An indigenous biosurfactant producing Gram-negative-rod-shaped bacterial strain (KISRQC) was isolated from oil-contaminated soil in Kuwait. The strain was able to reduce the surface tension below 41.1 mN/m with all tested hydrocarbons and had excellent emulsification activity toward crude oil. The strain was identified as Burkholderia cepacia. Crude biosurfactant extract from this strain was found to contain lipid with 94.9% unsaturated fatty acids. The crude biosurfactant extract was able to reduce the surface tension to 24.6 mN/m at concentration 2 g/l. Characterization of the crude biosurfactant extract activities indicated that it was stable when exposed to high and low temperatures for a reasonable time period (>month) and in the presence of 5% NaCl. The crude biosurfactant extract was effective in recovering 99% of the residual crude oil from oil-saturated sand packs. This study shows the ability of Burkholderia cepacia KISRQC to produce a highly stable biosurfactant with the strong emulsification ability towards crude oil from non-toxic material. Suggesting its potential for commercial exploitation for enhanced bioremediation of oil-contaminated soil and oil recovery from the soil. *Corresponding authors: Qadreyah Almatawah, Kuwait Institute for Scientific Research, Environment and Life Sciences Research Center, PO Box 24885 Safat, 13109, Kuwait, Tel: 0096524989116; Fax: 0096524956659; E-mail: qmutawa@kisr.edu.kw Received November 13, 2017; Accepted November 17, 2017; Published November 23, 2017 Citation: Almatawah Q (2017) An Indigenous Biosurfactant Producing Burkholderia cepacia with High Emulsification Potential towards Crude Oil. J Environ Anal Toxicol 7: 528. doi: 10.4172/2161-0525.1000528 Copyright: © 2017 Almatawah Q. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Almatawah Q (2017) An Indigenous Biosurfactant Producing Burkholderia cepacia with High Emulsification Potential towards Crude Oil. J Environ Anal Toxicol 7: 528. doi: 10.4172/2161-0525.1000528


Introduction
Burkholderia cepacia, earlier known as Pseudomonas cepacia, is a motile aerobic oxidase positive Gram-negative bacillus commonly found in moist environments and liquid reservoirs. The strain was first described in 1950 by Burkholder [1] as the cause of soft rot of onions. B. cepacia is now gaining increasing interest in agriculture, biotechnology, and medicine. The reason for this particular interest includes the organism's abilities to promote plant growth by antagonizing soil borne plant pathogens [2][3][4]; to degrade hydrocarbons and thus contribute in the bioremediation of contaminated soil and water [5,6]; and to cause opportunistic human infections, mostly in patients with chronic granulomatous disease [7,8] and cystic fibrosis (CF) [9][10][11][12].
B. cepacia can degrade a wide variety of compounds as carbon and energy sources [13], including a variety of aromatic compounds [14,15] pesticides and herbicides [16]. It has the ability to degrade benzo(a) pyrene and other fused ring compounds [17], and biodegradation of heavy crude oil [18]. The organism had shown remarkable potential as an agent for environmental pollution bioremediation.
Biosurfactants are amphiphilic compounds produced on living surfaces frequently on microbial cell surfaces or excreted extracellularly with noticeable surface and emulsifying activities and have been described as anti-adhesive antimicrobial agents [19,20]. They contain hydrophilic and hydrophobic molecules that confer the ability to accumulate between fluid phases, hence reducing surface and interfacial tension at the surface and interface respectively [19,21]. Biosurfactants exhibit a broad variety of chemical structures such as lipopeptides and lipoproteins, glycolipids, lipopolysaccharides, phospholipids, fatty acids, and polymeric lipids [22,23]. In recent years, the importance of biosurfactants has increased remarkably as they became potential candidates for many marketing applications in the petroleum, pharmaceuticals, biomedical, and food processing industries [24]. They have many advantages over chemical surfactants including lower toxicity and higher biodegradability, healthier environmental compatibility, high selectivity, and effectiveness at extreme temperatures, salinities or pH values [24][25][26][27]. In the present study, the isolation of a potent biosurfactant-producing bacterium, Burkholderia cepacia strain KISRQC with potential value in microbial enhanced oil recovery is reported. The effects of carbon sources on biosurfactant production and the biosurfactant chemical nature were also investigated. Furthermore, Microbial Enhanced Oil Recovery (MEOR) suitability tests and stability of the biosurfactant to environmental stresses are presented in this study.

Bacteria and growth conditions
The microorganism used in this study, code name KISRQC, is a Gram-negative-rod-shaped bacterium isolated from oil-contaminated soil in Kuwait by previously described method [28]. The strain was identified as Burkholderia cepacia by MIDI Labs laboratories (USA) using 16S rRNA gene sequence similarity, which was performed using a Perkin-Elmer Applied Biosystem's MicroSeq TM microbial analysis software and database. The sequence determined for the 16S rRNA gene resulted in 99.7% match with Burkholderia cepacia (Gene Bank accession no. KY047601). The strain was grown on minimal media containing (g/l distilled water): Na 2

Carbon source effect determination
To determine the effect of the carbon source on biosurfactant production, KISRQC strain was grown in minimal media containing 3% yeast extract and 3% olive oil and incubated at 37°C for 24 h. The 24 h culture was used to inoculate minimal media containing 3% yeast extract and different carbon source (2% crude oil; 1% diesel; 2% hexadecane; 1% kerosene; 3% olive oil; 3% paraffin; 1% xylene) and incubated for 96 h at 37°C. The growth was observed as an increase in absorbance at 660 nm and converted to g/l based on a predetermined calibration curve. After the incubation period, the broths surface tensions, emulsion indexes, and cell concentrations were measured.

Surface activity measurement
Surface tension and critical micelle concentration (CMC) were measured by the Du Nouy ring method [29] using Kruess K10T tensiometer (Kruess, Optische-Mechanische Werlostatten, Hamburg, W. Germany) with a 6 cm diameter platinum-iridium ring. The CMC is defined as the surfactant concentration necessary to initiate micelle formation. There will be no further decrease in the surface tension upon reaching the CMC when anadditional surfactant is present. CMC was determined by diluting the whole culture broth and measuring the surface tension of each dilution [30,31]. The reciprocal of CMC is proportional to the total surface-active compound amount present in the solution and can be used as an approximate measure of biosurfactant concentration.

Determination of emulsification activity
The emulsification activity of the biosurfactant was determined by the addition of 6 ml hexadecane to 4 ml of the test broth or biosurfactant in a 15-ml graduated tube. The mixture was mixed vigorously for 2 min and allowed to stand for 24 h at room temperature. The emulsification index (E 24 ) was calculated by dividing the measured height of the emulsion layer by the total height of the mixture and multiplying it by 100 [29,32,33].

Dry cell weight measurement
Biomass dry weights were determined by measuring the culture optical density at 660 nm, and then centrifugation of 100 ml sample of the culture broths at 10,000 xg for 10 min. The pellet was washed twice with distilled water, re-suspended in 10 ml of distilled water, filtered through a pre-washed and weighed 7-cm-diameter Whatman No.1 filter paper, placed on a pre-weighed aluminum dish, dried at 105°C overnight, and reweighed after 24 h. A standard curve was constructed from the dry cell weights and their corresponding OD 660 values.

Biosurfactant extraction
Cells in culture broth were disrupted by sonication for 15 min using an MSE 150 W ultrasonic disintegrator at 14 amplitude and centrifuged at 15,000 xg for 15 min at 0°C. The hydrophobic layer located at the surface was extracted using the methyl tertiary-butyl ether (MTBE)chloroform (1:1) solvent system. The solvent layer was separated from the aqueous phase and removed by rotary evaporation at 50°C under reduced pressure [34]. The resulting crude extract was stored at -20°C for later analysis.

GC analysis
The fatty acid composition of the crude biosurfactant extracts was analyzed by Hewlett Packard HP5890 gas chromatograph, equipped with a flame ionization detector and a capillary column fused with methyl silicone (50 m length, 0.25 mm internal diameter). The operating temperature of the detector was 300°C, and that of the injector was 280°C. The column temperature was set at 120°C for the first 5 min, then increased to 200°C at a rate of 5°C/min and then increased again to 225°C at the rate of 4°C/ min until the final time (35 min).

Stability studies
The effects of several environmental parameters on the surface activity of the biosurfactant were determined. NaCl stability was determined by dissolving the crude biosurfactant in 5% NaCl (200 µl/25 ml), and the surface tension was then measured. To determine the heat stability of the surface active compounds, the culture broth, the culture supernatant, the cell suspension, and the biosurfactant crude extract (200 µl in 25 ml 0.9% NaCl) were heated at 100°C for 15 min and allowed to cool to room temperature, and the surface tension was then measured and compared to the corresponding values before heat treatment. Time stability was measured by incubating the biosurfactant crude extract at two different temperatures for five weeks, and the surface tension was then measured at one week intervals and compared to the corresponding values.

Sandpack test
A glass column (50 ml volume) was packed with acid washed sand (25-60 mesh 30 g). The column was then saturated with 6 ml crude oil and flooded with pore volumes of water until no further oil was released. The remaining residual oil was flooded with crude biosurfactant as a continuous flood of at least three pore volumes and released residual oil percentage was measured. Table 1 showed the effect of carbon source on biosurfactant production. There were significant differences between the control and other tested carbon sources in the surface tension value. The surface tensions were below 41.1 mN/m for all tested carbon sources. Surface tension decreased from 41.1 to 27 mN/m, with the greatest reduction (34%) observed in culture enriched with 3% olive oil. Moreover, cell concentration was increased from 2.1 g/l to 20.4 g/l with olive oil enriched culture. Crude oil also showed an increase in cell concentration from 2.1 g/l to 17.1 g/l. The best emulsion index was 70% when olive oil used as carbon source. The ability of culture broth enriched with 3% olive oil to emulsify crude oil was tested. The broth was found to be able to emulsify crude oil (Figure 1).

Effect of incubation period on biosurfactant production
The surface tension was monitored during the growth cycle of KISRQC strain to determine the optimum incubation period. The surface tension in the culture medium was found to be proportional to culture turbidity and biomass production ( Figure 2). The reduction of culture surface tension occurred during the exponential growth in batch culture. The lowest value of surface tension (26.8 ± 0.1 mN/m) was recorded 96 h after culture inoculation.

Biosurfactant isolation
To estimate the effectiveness of the biosurfactant concentrates, samples of culture broth were taken at the logarithmic phase of growth (48 hr) and extracted according to the method described earlier. The concentrates obtained were re-dissolved in 0.9% NaCl, and the surface tensions for a series of dilutions of them were measured (Figure 3). The biosurfactant crude extract was found to reduce the surface tension to 24.6 ± 0.1 mN/m at a concentration of 2 g/l.

Biosurfactant characterization
The crude biosurfactant produced was characterized according to five criteria: the capacity to reduce surface tension, heat stability, NaCl stability, time stability, and the ability to mobilize oil from sand pack. The isolated crude biosurfactant was able to reduce the surface tension to 24.6 ± 0.1 mN/m. Table 3 shows the results of experiments on the effect of heat treatment on the biosurfactant activity for KISRQC culture. No significant change in biosurfactant activity occurred when culture broth, supernatant, cells, and biosurfactant crude extract were exposed to heat treatment. The properties of the biosurfactant present in all tested samples, i.e., surface tension, remained stable after exposure to high temperatures of 100°C for 15 min.
Experimental results on the effect of 5% NaCl on the surface tension and the emulsification ability of the biosurfactant on crude oil showed no significant change of the surface tension value (27.1 ± 0.6 mN/m) and emulsification activity (Figure 4). The crude biosurfactant reduced 62.3% of the 5% NaCl solution original surface tension. The biosurfactant properties remained stable for a reasonable time period under low temperature (4°C) and high temperature (45°C). After five weeks, the crude biosurfactant was able to reduce the surface tensions to 28.6 ± 0.1 mN/m and 28.3 ± 0.2 mN/m at 4°C and 45°C respectively. In sand pack experiments, the crude biosurfactant was found to be able to elute nearly 99% of the crude oil from the sand packs ( Figure 5).

Discussion
In recent years, several studies have shown that a number of Burkholderia species such as Burkholderia thailandensis [34,35], Burkholderia glumae [36], Burkholderia kururiensis [37], Burkholderia plantarii [38], and Burkholderia gladioli [39] were able to produce biosurfactant. However, the ability of Burkholderia cepacia to produce a biosurfactant was not sufficiently documented in the literature. Burkholderia cepacia was reported as one of the most predominant culturable species in crude oil and for their ability to degrade both aliphatic and aromatic hydrocarbons [40]. Limited studies have focused on the production of biosurfactant by Burkholderia cepacia. One study, show the inability of Burkholderia cepacia to produce a biosurfactant [41] and other studies showed its ability to produce a biosurfactant [42,43]. A study on the bioavailability of non-aqueous phase liquids (NAPLs) for Burkholderia cepacia, provided no evidence for the release of extracellular boisurfactants [41]. The study interrelated the release of lipopolysaccharides (LPS) in culture media for enhancement of cell surface hydrophobicity and adherence affinity to NAPLs. Another study reported the ability of Burkholderia cepacia MFW3 to produce biosurfactant when grown on whey waste water [42]. The biosurfactant was characterized as lipopeptide structure and reduced the surface tension by 12%, with maximum emulsification activity of 59% with mineral oil. Moreover, a study also reported the ability of Burkholderia cepacia to produce an active surface agent [43]. In this study, the ability of Burkholderia cepacia KISRQC to produce an active biosurfactant is documented. The biosurfactant reduced the surface tension by 34%, with maximum emulsification activity of 70% with hexadecane. Which is better than the previously reported results [42]. Furthermore, olive oil was found the best carbon source for the biosurfactant production. Pseudomonas fluorescens Migula 1895-DSMZ was reported to produce rhamnolipid biosurfactant with the highest yield when olive oil used as a carbon source [44]. Plant-derived oils have been documented as an excellent carbon substrates for biosurfactant production by Pseudomonas aeruginosa [45,46]. The Burkholderia gladioli culture produced most biosurfactant when enriched with 5% corn oil [39]. Burkholderia thailandensis produced a series of long chain rhamnolipids when grown in canola oil [35]. Although, diesel and hexadecane was also found to enhance biosurfactant production by Burkholderia cepacia KISRQC, one study reported the opposite [41]. This may be related to the application of different growth conditions. The production of Burkholderia cepacia KISRQC biosurfactant is 2 g/l when grown at 37°C. This is nearly similar to the production of Burkholderia thailandensis E264 biosurfactant, 1.99 g/l biosurfactant when grown at 30°C [35]. Burkholderia thailandensis E264 culture grown at 30°C generated a dry cell biomass throughout the growth period with 7.71 g/l after 264 h, whereas Burkholderia cepacia KISRQC culture grown at 37°C generated a dry cell biomass throughout the growth period with 7.7 g/l after 48 h. Burkholderia thailandensis E264 was found to produce di-rhamnolipids with C14-C14 chain length fatty acid moiety in most abundance (41.88%) and others contained chains ranging from C10-C12 to C16-C16 chain length [34]. The potential of the long-chain rhamnolipids produced by Burkholderia species for lowering surface tension and decreasing the CMC was demonstrated [34]. Sophorolipids, a class of biosurfactants produced by Candida bombicola, was correlated it's decreasing of the CMC to carbon chain length. The additional CH2 groups were found to render the molecule more hydrophobic and facilitate micelle formation [47]. The ability of Pseudomonas aeruginosa to reduce the surface tension to 28.1 mK/m with maximum emulsification activity of 70% in culture medium enriched with 2% corn oil was reported [39]. Whereas, Burkholderia gladioli was found to reduce the surface tension to 45.4 mK/m with maximum emulsification activity of 69% in culture medium enriched with 5% corn oil. In this study, we reported the ability of Burkholderia cepacia KISRQC to reduce the surface tension to 27 mK/m with maximum emulsification activity of 70% in culture medium enriched with 3% olive oil. Most of the reported biosurfactant from Burkholderia species were rhamnolipids that have long alkyl chains than those produced by Pseudomonas aeruginosa [48]. Interestingly, Burkholderia cepacia KISRQC biosurfactant crude extract was found to contain 14.4 g/l lipid with C18 unsaturated fatty acids in the highest abundance (94.9%) followed by C17 (5%), C16 (0.32%) and C14 (0.12%). The highest abundance of C18 fatty acid in Burkholderia cepacia KISRQC biosurfactant means that the biosurfactant structure is different from the reported biosurfactant from Burkholderia species.   The isolated crude biosurfactant was able to reduce the surface tension, stable after exposure to high temperatures of 100°C, and when incubated at 4°C and 45°C for five weeks. The production of biosurfactant was in the exponential growth phase, which indicates that the production of biosurfactant is a primary product of metabolism and suggests that it would be possible to be produced effectively in a continuous process. Since Kuwaiti soil contains high concentrations of sodium chloride and other salts, it is essential that a biosurfactant should be effective under these conditions. The surface tension and the emulsification ability of the biosurfactant on crude oil was found stable at 5% NaCl. Moreover, the crude biosurfactant extract was effective in recovering 99% of the residual crude oil from oil-saturated sand packs. Several researchers have been using sand pack columns for evaluation of surfactant suitability for enhanced oil recovery [33,49,50].
So far, biosurfactants used has been limited due to the high cost of raw materials and processing. An inexpensive hydrophobic materials sources like vegetable oils or waste cooking oil and carbohydrates is a good approach for reducing industrial waste generation [51]. B. cepacia KISRQC seems a promising strain for large-scale biosurfactant production since it can grow on no toxic material such as olive oil; which is an advantage from an industrial point of view. Moreover, its biosurfactant showed a strong emulsification ability toward crude oil, suggesting its potential for commercial exploitation for enhanced bioremediation of oil-contaminated soil and oil recovery from the soil.