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ISSN:2157-7463
Journal of Petroleum & Environmental Biotechnology
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A Potent Biosurfactant Producing Bacterial Strain for Application in Enhanced Oil Recovery Applications

Gamil A. Amin*
Taif University, Taif, Saudi Arabia
Corresponding Author : Dr. Gamil A. Amin
Taif University, Taif, Saudi Arabia
Email: [email protected]
Received August 16, 2010; Accepted November 24, 2010; Published November 26, 2010
Citation: Amin GA (2010) A Potent Biosurfactant Producing Bacterial Strain for Application in Enhanced Oil Recovery Applications. J Pet Environ Biotechnol 1:104. doi:10.4172/2157-7463.1000104
Copyright: ©2010 Amin GA. 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.

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Abstract

The capability of hydrocarbon utilizing bacteria to produce biosurfantants was investigated. Out of 38 spore forming alkane utilizing bacteria isolated from Jeddah Refinery facilities, a potent surfactin producing bacterium was isolated, by enrichment technique. The isolate was purified and partially characterized as members of the genus Bacillus and was designated as BDCC-TUSA-3. It was able to biodegrade 11.0 g of n-hexadecane and consume more than 82% in 55 h, but surfactin was not detected. When grown on Maldex-15, a cheap by-product recovered during manufacturing of high fructose syrup from corn starch, up to 4650 mg.l -1 of surfactin was produced in 40 h. The kinetic characterization, for cell growth and surfactin production from Maldex-15 was determined. A maximum specific growth rate of 0.462 h -1 , conversion efficiency of 98.8% and volumetric reactor productivity of 155 g surfactin .l -1 .h -1 were achieved. The obtained results suggest that BDCC-TUSA-3 strain can be of great importance as a feasible MEOR agent particularly in nearly exhausted oil fields.

Keywords
Surfactin; Bacillus; Biodegradation; Biosurfactants; MEOR
Introduction
Recently, attention has been given to microbial enhancing oil recovery (MEOR) processes. Biosurfactants produced by certain microorganisms are among the most promising MEOR agents [1]. They enhance the recovery of oil by reducing the interfacial tension (IFT) between the oil and water interfaces, or by mediating changes in the wettability index of the system. Microbial surfactants have several advantages over chemical surfactants such as lower toxicity, higher biodegradability and effectiveness at extreme temperatures or pH values [2]. Numerous reviews are available on the production and application of biosurfactants [1,3,4]. Many of the potential applications that have been considered for biosurfactants depend on nature of the biosurfactant and whether it can be cost-effectively produced.
Surfactin is one of the most powerful biosurfactants produced by various strains of Bacillus subtilis, a gram-positive, motile, spore forming bacteria [5,6,7]. It is composed of a seven amino-acid ring structure coupled to a fatty-acid chain via lactone linkage. Surfactin lowers the surface tension from 72 to 27.9 mN/m at concentrations as low as 0.005% and interfacial tension of water/hexadecane to <1 mN/m [8].
The present study is aimed at finding a bacterial strain that could effectively produce surfactin from inexpensive waste substrate and could survive simultaneously at harsh conditions encountered in oil well tubing.
Materials and Methods
Sampling site
Sediment samples were collected, at summer time, max. temp 45oC, from one oil transportation pipe at a repair and maintenance site, Jeddah Oil Refinery, South Jeddah, West of Saudi Arabia.
Enrichment procedure
In order to selectively enrich thermophilic microbial strains that could degrade hydrocarbons and hopefully produce biosurfactant, a chemostat culture was set in a 2-l glass vessel with a working volume of 1 l. minimum salt medium (MSM) was added at the following composition: NaCl (0.001%), MgSO4 (0.06%), CaCl2 (0.004%), FeSO4 (0.002%) and 0.1 ml of trace element solution containing (g.l-1) 2.32 ZnSO4.7H2O, 1.78 MnSO4.4H2O, 0.56 H3BO3, 1.0 CuSO4.5H2O, 0.39 NaMoO4.2H2O, 0.42 CoCl2.6H2O, 1.0 EDTA, 0.004 NiCl2.6H2O and 0.66 Kl. The MSM was additionally supplied with n-hexadecane as the sole carbon and energy source at 11.0 g.l-1 and inoculated with sediment sample at 5% (w/v). Temperature and pH were controlled at 55°C and 7.2 respectively and agitation speed was maintained at 200 rpm and cultivation continued for 5 days. This was repeated 8 times after which serial dilutions of the obtained culture were prepared and plated on MSM agar plates with n-hexadecane at 11.0 g.1-1 as carbon and energy source [9]. The microbial colonies thus developed were further tested, purified and sub-cultured on the same medium. A frozen stock cultures were stored in 30% glycerol at -70°C.
Biomass (dry weight): Bacterial biomass was determined by centrifuging a known volume of fermentation broth at 1000g for 15 min. The biomass was washed twice with distilled water and dried overnight at 90°C to constant weight.
Growth characteristics of bacterial isolates: Vegetative cell morphology observations were made on cultures grown for 24 hr, on nutrient agar (NA) medium, at 30°C. Microorganisms were examined at 1000 x magnification, using phase contrast microscopy, for shape of cells, presence of chains, and for reaction with gram stain [10]. To determine motility, strains were grown on slopes of NA and after 6 h, or as soon as growth appeared thereafter, a loopful of the liquid at the base of the slope was examined at 1000 x magnification by phase contrast microscopy.
The catalase activity of bacterial isolates was detected by resuspension of a colony in a 3% solution of hydrogen peroxide (Sigma).
The ability to grow on different carbon sources was determined by monitoring the optical density at 600 nm of cultures grown in nutrient broth supplied with glucose, xylose, maltose, sucrose, cellobiose, galactose, starch, mannitol or Tween 80 as carbon source.
Biosurfactant production: In order to test for biosurfactant production, 500 ml shake flasks each charged with 250 ml of MSM containing 20 g.l-1 of Maldex-15 as carbon source were used. Maldex-15 is an α-amylase dextrin produced during the commercial production of high fructose syrup from corn starch. It contains 1% glucose, 3% maltose, 5% maltotriose and 91% of oligo- and megalosaccharides [11]. The flasks were inoculated with a 24 h culture of each bacterial strain at 5% (v/v) and incubated in a rotary shaker at 50°C and 200 rpm. Samples were taken at the end of each cultivation run, centrifuged at 1000g for 15 min and examined for biosurfactants.
Screening for biosurfactant activity: Biosurfactant activity of bacterial isolates was detected by using oil spreading technique and emulsification stability test in three different oils: kerosene, crude oil and motor oil.
Oil spreading technique: The capabilities of the bacterial isolates were determined by measuring of the diameter of the clear zones occurred when a drop of a biosurfactant-containg solution is placed on an oil-water surface [12]. A 50 ml of distilled water was added to a Petri dish, 15 cm diameter, followed by the addition of 20µl of oil to the surface of water. Then, 10µl of supernatant of bacterial culture was added. The diameter of the developed clear zones was determined in triplicate.
Emulsification stability (E24) Test: E24 of culture samples was determined by adding 2 ml of oil to the same amount of culture, mixing with a vortex for 2 min and leaving to stand for 24 hours. The E24 index is given as percentage of height of emulsified layer (mm) divided by total height of the liquid column (mm) [13].
Surface tension measurements: They were made with a Fisher Autotensiomat (Fisher Scientific Co., Pittsburgh, Pa.). Relative surfactin concentrations were determined by diluting the broth until the critical micelle concentration (CMC) was attained [14].
Isolation of surfactin: The method described by Cooper et al. [15] was used. Crude surfactin was isolated by adding concentrated hydrochloric acid to culture broth after removing biomass by centrifugation. A precipitate formed by decreasing the pH to 2 which could be collected, dried and extracted with dichloromethane. The solvent was removed under reduced pressure to give an off-white solid. Further purification was achieved by recrystallization. The dichloromethane extract was dissolved in distilled water containing sufficient NaOH to give pH 7. This solution was filtered through Whatman no. 4 paper and reduced to pH 2 with concentrated HCl. The white solid was collected as pellets after centrifugation.
Qualitative characterization of the produced biosurfactant: Thin Layer Chromatography was used in order to characterize biosurfactant produced in culture broth of bacterial isolates according to the method of Yin et al. [16]. A portion of the crude biosurfactant was separated on a silica gel plate using CHCl:CHOH:H2O (70:10:0.5, v/v/v) as developing solvent system with different color developing reagents. Ninhydrin reagent (0.5 g ninhydrin in 100 ml anhydrous acetone) was used to detect surfactin as red spots.
Time course for cell growth and biosurfactant production: In another set of experiments, a chemostat culture of 3-l charged with 1700 ml of MSM containing 40 g.l-1 of Maldex-15 was conducted for each of the selected bacterial strains. Cultivation conditions were set as described above. Samples were taken at regular intervals and analyzed for biomass, surfactin and residual carbohydrates.
Analysis
The total carbohydrate in cultivation broth was estimated by the phenol-sulfuric acid method using mannose as a standard [17]. The specific growth rate was determined according to Pirt [18]. The following equation was used.
µ.loge.t = log X2- log X1
Where µ = specific growth rate,
loge = natural Logarithmic,
X1 and X2 = Two values for biomass (DW) concentrations at mid-Logarithmic phase of cell growth and t= time between measurements of X1 and X2.
Results
Isolation and partial identification of hydrocarbon utilizing and biosurfactant producing microorganisms
Forty eight microbial strains with the ability to grow on n-hexadecane as sole carbon and energy source and representing the different colony morphologies were obtained (Figure 1). These include 38 (9.2%) isolates as spore-forming bacteria, 8 (16.7%) as nonspore forming bacteria and 2 (4.2%) as yeasts. Non-spore forming bacteria and yeast strains were not further used. This study was therefore limited to biosurfactant production by the spore forming alkane utilizing bacteria.
Out of the 38 spore forming bacterial isolates, 3 showed effective biodegradation of hydrocarbon with more than 69% conversion efficiency. The three bacterial isolates were named after the Biotechnology Department Culture Collection, Taif University, Saudi Arabia as BDCC-TUSA-1, BDCC-TUSA-2 and BDCC-TUSA-3 and were used for the biosurfactant production experiments.
Morphological and biochemical characteristics
The morphologically distinct isolates were identified by morphological, physiological, and chemo-taxonomical properties in accordance with Bergy’s Manual of Determinative Bacteriology [19].
The three selected isolates were spore formers, aerobic, motile, gram positive rods, catalase positive, indol negative, thermophilic (growing vigorously at 55°C) and can utilize glucose, xylose, maltose, sucrose, cellobiose, galactose, starch and mannitol as carbon and energy sources but they cannot utilize Tween 80. This partial identification characterizes them as members of the genus Bacillus [20]. These results are in good agreement with those obtained by Pokethitiyook et al. [21] and Sadeghazad and Ghaemi [22] who reported that Bacilli species were among the most effective microorganisms in the biodegradation of alkane and the prevention of wax precipitation and deposition.
Examination for biosurfactant production
Oil displacement capability and emulsification activity are among the most important characteristics of biosurfactant producing microorganisms [1,7]. These characteristics have been shown to increase the biodegradation rate of hydrocarbons, stimulate microbial growth, decrease crude oil viscosity and enhance oil recovery at oil fields [23-25]
In the present study, biosurfactants were never detected in culture broth of bacterial isolates grown in MSM in presence of n-hexadecane as the sole carbon source. On the contrary, Margaritis et al. [23] and Etoumi [26] found various types of biosurfactants with bacterial strains of Pseudomonas and Actinomycetes species when grown on n-hexadecane. This discrepancy is most probably due to nature of microorganisms and fermentative substrates utilized.
Therefore, it was attempted to test possibilities for biosurfactant production by these isolates in MSM supplies with Maldex-15 as a fermentative substrate. The three selected bacterial isolates with the highest utilization rate of alkane (Table 1) were tested for biosurfactant production from an alternative carbon source as described in the Materials and Methods section. When growing in MSM with Maldex-15 as the carbon source, theisolates produced biosurfactant with varying concentration as culture supernatants exhibited variable displacement capabilities (Table 2) and emulsification activities (Table 3). Cleary, isolate BDCC-TUSA-3 showed better performance compared to the other two isolates. An oil displacement area, emulsification index (E24), with crude oil, of 67.9 cm2 and 97% were recorded for isolate BDCC-TUSA-3 compared to only 12.6 and 28% for isolate BDCC-TUSA-2 and 32.2 and 89% for isolates BDCC-TUSA-1 respectively. These results are comparable with those reported by Etoumi [26] and Margaritis et al. [23] using Pseudomonas species.
While the emulsification indices recorded for the three hydrocarbons, kerosene, crude oil and motor oil (Table 3), there was a difference in emulsification activity of isolate BDCC-TUSA-3 on the three hydrocarbons. An emulsification index of 97% was achieved with crude oil, 90% for motor oil and 86% for kerosene. Thus, isolate BDCC-TUSA-3 showed a preferential emulsification in the order of crude oil>motor oil>kerosene.
Isolation and identification of biosurfactant
The produced biosurfactant was identified as surfactin by TLC technique as red color spots were developed upon application of ninhydrin reagent. Again, isolate BDCC-TUSA-3 exhibited the highest Rf value and proved to be the best surfactin producers among tested isolates (Table 4).
Time course for cell growth and biosurfactant production
Figure 2 and Table 5 show the growth pattern and surfactin production from Maldex-15 by the three selected bacterial isolates. As evidenced from (Figure 2), Madex-15 supported the growth and surfactin production of all isolates but at varying rates and efficiencies with isolate BDCC-TUSA-3 being the best. Almost no lag phase was observed during the growth. About 20% of carbohydrate was consumed during the first 10 h of cultivation with 0.8-1.2 g.l-1 of bacterial biomass. No surfactin was detected during this period. With isolate BDCC-TUSA-3, maximum biomass concentration of 5.96 g.l-1 was achieved after 20 h followed by the maximum production of surfactin of 4650 mg.l-1 at 30 h with almost complete consumption of Maldex-15 (98.7%?) (Table 5). This is in agreement with Bala et al. [27] and Noah et al. [6] but disagree with Sheppard et al. [28] who reported the maximum surfactin production in mid log phase and Gong et al. [7] who found that surfactin production was a growth associated process. This discrepancy could be attributed to nature of the microorganisms and environmental and cultural parameters employed in each study.
Isolates BDCC-TUSA-1 (Figure 2A) and BDCC-TUSA-2 (Figure 2B),resulted in much lower Maldex-15 conversion efficiencies (68.1 and 56.9 % respectively) with biomass concentrations of only 2.82 and 1.47 g.l-1 respectively. At the same time, the bacterial isolate BDCC-TUSA-1 produced only 2050 mg.l- of surfactin even when its capacity to degrade n-hexadecane was the best (Table 1). The lowest surfactin production of 560 mg.l-1 was recorded for isolate BDCCTUSA- 2 (Table 2).
As illustrated in (Figure 2), a gradual decrease in surface tension was observed for the three bacterial isolates. This was coordinated with the onset of surfactin production. The minimum surface tension was detected after about 15 h and maintained throughout the remaining cultivation period. In contrast, surfactin production continued to rise reaching its maximum at almost 25 h, where stationary phase of cell growth has already been reached.
With respect to overall kinetic parameters, isolate BDCCTUSA- 3 showed advantage over the other isolates (Table 2). This isolate achieved the highest values: specific growth rate 0.462 h-1, growth yield 0.151 gcells.gMaldex-15, surfactin yield 117.9 gsurfactin/gMaldex-15, surfactin yield by unit of dry weight 780.2 mgsurfactin/gcells and the highest volumetric reactor productivity of 155 g.l-1.h-1. Noah et al. [6] developed a process for continuous surfactin production using an airlift bioreactor utilizing Bacillus subtilis and recording one of the highest specific growth rates, reported in literature (0.447h-1) which is lower than that reported in this study . This is most probably due to the increased concentrations of free glucose and short-chain maltodextrins present in Maldex-15 which might initiate active cell growth and multiplication.
Over the last 50 years, a number of promising results have been obtained in improving the yield of surfactin by fermentation. Arima et al. [29] firstly produced surfactin, with an output with only 50–100 mg.l-1 in a 24- -h culture. Then, Cooper et al. [15] developed a process with continuous product removal and metal cation addition which resulted in improved surfactin yield of 780 mg.l-1. Mulligan et al. [30] used the ultraviolet mutant of Bacillus subtilis ATCC 21332 which produced over three times more surfactin (1124 mg/L). More recently, Sen and Swaminathan [31] optimized the fermentation medium and obtained a maximum surfactin production of 760 mg.l-1. Cleary, the results obtained in the present study?? Using isolate BDCCTUSA- 3 compare very favorably with those reported in literature and demonstrate its superiority. As a rapid grower on alkanes, motile and effective producer of surfactin, the bacterial isolate BDCC-TUSA-3 has a potential applications as MEOR agent in nearly exhausted oil fields and/or those with history of wax paraffin deposition.
Conclusion
The present study provides a remarkable thermophilic bacterial strain with a promising potentiality in the use as MOER agents. The Maldex-15, a by-product recovered during manufacturing of high fructose syrup from corn starch, was proved to be a viable fermentative substrate for relatively high production of surfactin.
Beside availability of the potent BDCC-TUSA-3 strain and the cheap fermentative substrate for cell growth and production of surfactin, surfactin production process needs to be fully optimized at both biological and engineering level in order to compete successfully with chemical surfactants. Work isin progress in order to optimize cultural and environmental conditions required for higher rates for substrate utilization and surfactin production as well.
Acknowledgement
The authors are grateful to Department of Academic Affairs, Taif University, K.S.A. for the financial support for this work.
References
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