Emulsification Properties of Bacterial Biosurfactants Native to the Yellow River Delta on Hexadecane and Diesel Oil

Kevin B. Cheng a,c, Zhao Jian b,d, Dr. Zhenyu Wang b,e
b

Department of Biological Sciences, Clarkson University, Potsdam, NY 13699, USA College of Environmental Sciences and Engineering, Ocean University of China, Qingdao, Shandong Province, 266100, Peoples Republic of China c Intern, kevinbcheng@gmail.com, d Graduate Advisor, zhaojian047@126.com, e Mentor, wang0628@ouc.edu.cn Draft Completed 25-July 2008

Abstract
The Yellow River Delta (YRD) is an important agricultural and ecological base that is being threatened by crude oil contamination. The remediation of the YRD can be potentially assisted by biosurfactants. Three native unknown biosurfactant-producing bacteria have been isolated (L1, L2, L3). In this paper, we measured the emulsification properties of the three biosurfactants in terms of emulsification activity (EA) and emulsification stability (ES) and compared them to two artificial surfactants, sodium dodecyl sulfate (SDS) and Triton X-100. We found the biosurfactants had comparable EA and superior ES to the two artificial surfactants.

1. Background
With its close proximity to Shengli Field, Chinas second largest proven oilfield, the Yellow River Delta (YRD) is at high risk for crude oil contamination [1, 2]. The faltering integrity of the YRD has grave environmental consequences. In addition to being a large source of petroleum, the YRD is a habitat for endangered migratory birds as well as an important agricultural base of China [1]. Therefore, the remediation of the YRD from crude oil is needed to protect a vital food source and habitat. Due to the heavy crude oil contamination of the Yellow River Delta and the lack of a totally effective clean up program, the remediation of the delta is of high importance. Bioremediation is the degradation of toxic pollutants through biological means. Biosurfactants are attractive for bioremediation because they are biodegradable and relatively non-toxic, making it an attractive compound to be released in bulk at a remediation site [3, 4]. Biosurfactants act by emulsifying hydrocarbons, increasing the solubilization of crude oil and subsequent availability for microbial degradation [5, 6]. Previously, the Wang lab investigated the degradation of hydrocarbon bacteria native to the YRD [7]. In this paper, crude oil was substituted for hexadecane and diesel oil. Substitution was necessary since the composition of crude oil is highly variable. Crude oil is mostly composed of hydrocarbon alkanes, aromatics and heteroaromatic compounds containing sulfur or nitrogen [8]. Hexadecane is an alkane, a class of hydrocarbons of which is a large component of crude oil while diesel oil has also been cited as a good experimental substitute for crude oil [4]. Emulsification properties of surfactants were measured in terms of emulsification activity (EA) and emulsification stability (ES) [9]. EA is the ability for a surfactant to form an emulsion under given

conditions and is directly related to oil droplet size, the smaller the size the greater the activity. By nature, emulsions are thermodynamically unstable and eventually separate into oil and water phases [10]. Therefore, ES is the measure of how slowly an emulsion separates into oil and water phases.

2. Introduction
The Yellow River Delta is an important agricultural and ecological base that is being threatened by crude oil contamination. The remediation of the YRD can be potentially assisted by biosurfactants, biodegradable and relatively non-toxic compounds that increase the uptake of hydrocarbon-degrading bacteria. The Wang lab has isolated three unknown biosurfactant-producing (BP) strains of bacteria from the YRD (named L1, L2 and L3). The purpose of this research is to examine the biosurfactant extracted the three BP strains. In this paper, we measured the emulsification properties of the biosurfactants in terms of emulsification activity and stability with two crude oil substitutes, hexadecane and diesel oil. EA and ES of our biosurfactants were measured by turbidimetry. In an emulsion, the smaller the oil droplets the greater scattering of light [10]. As a result, we can measure EA by measuring absorbance using a spectrophotometer. ES is measured by measuring the absorbance over 60 minutes. For EA and ES, we measured the absorbance at 540nm [11]. This method is explained in detail in Section 3.4. We also compared our results with two artificial surfactants, sodium dodecyl sulfate (SDS), an anionic surfactant, and Triton X-100, a nonionic surfactant.

3. Materials and Methods

3.1 Chemicals

Hexadecane was purchased from Sigma-Aldrich, USA. Diesel Oil was purchased from a gas station close by to the OUC Laoshan Campus, China. Sodium Dodecyl Sulfate (SDS, 95%) was purchased from Beijing Solarbio Science and Technology Co., Ltd, China. Triton X-100 (99%) was purchased from Qingdao Huishi Trading Co., Ltd, China. All chemicals purchased except Diesel Oil were reagent grade.
3.2 Growth of the Biosurfactant-producing Bacteria

The three strains of BP bacteria (L1, L2, L3) were obtained from a culture maintained by the Wang lab, originally isolated from a petroleum contaminated region of the Yellow River Delta. The three strains were growth separately in a 5L Erlenmeyer flask containing 1L growth medium, composing of 1L H2O, 1ml hexadecane, 1g K2HPO4, 1g KH2PO4, 1g (NH4)2SO4, 15g NaCl, 0.2g MgSO4, 0.02g CaCl2 and 0.02g FeCl3. The bacteria were incubated at 30C for 3 days with 2min of light shaking every day.
3.3 Extraction of the Biosurfactants

The growth medium for each strain was centrifuged in 50ml test tubes at 6000rpm for 20min at room temperature to separate the bacteria from solution (Centrifuge: HC-3018R. USTC Chuangxin Co., Ltd. Zonkia Branch, China). After each spin, the supernatant was collected, which was acidified to pH 2 with HCl. The biosurfactant was extracted from the supernatant using two volumes of 500mL chloroform/ethanol (2:1) solution in a separatory funnel. The bottom layer was extracted and collected. The solvent was removed from the biosurfactant by rotary evaporation at a temperature below 40C (Rotary Evaporator: Eyela NVC200, Tokyo Rikakikai Co,.Ltd). At heat gun was used sparingly to evaporate any remaining solvent. The weight of each product was recorded and the biosurfactants were stored in at -10C overnight.

3.4 Measuring Emulsification Activity and Stability of Biosurfactants

The biosurfactants were taken out of the refrigerator and were heated to room temperature. Each biosurfactant was diluted to 1g/L H2O based on its solid yield [12]. The pH of each solution was adjusted to 7-8. The emulsification activity and stability was measured using a method used by Lee et al (2008)[11]. For each biosurfactant, 4ml of biosurfactant solution was diluted with 1ml substrate (hexadecane or diesel oil). The mixture was vigorously shaken in a vortex mixer for 2min. The mixture was let stand for 10min and the turbidity was measured at 540nm using a Spectrumlab 752s spectrophotometer (Lengguang Tech., China). The blank cuvette only contained H20.The emulsification activity was the measured absorbance. The absorbance was measured every 10 minutes for 60 minutes. The log of the absorbance was plotted over time and the emulsification stability was calculated as the slope of the graph. For the control test, the 4ml biosurfactant in the above protocol was replaced with 4ml H20.
3.5 Comparison with Artificial Surfactants

The emulsification activity and stability of two artificial surfactants, SDS and Triton X-100, were measured. Both surfactants were diluted to 1g/L by weight and was analyzed using the procedure in section 3.4 using hexadecane and diesel oil.

4. Results
4.1 Extraction of Biosurfactants

Following the culturing the bacteria for 3 days and extraction, the solid yield was 0.4009g (L1), 0.5119 (L2) and 0.5594 (L3). L1 and L2 was light yellow while L3 had a white appearance. When extracting all strains using the separatory funnels, the bottom layer contained the biosurfactant was extracted, having a clear appearance. The top layer contained impurities and were discarded, having a cloudy white appearance.
4.2 Emulsification Activity and Stability of Biosurfactants, SDS and Triton X-100

The EA of the three biosurfactants (L1, L2, L3), SDS and Triton X-100 in hexadecane or diesel oil were measured over several trails (Fig. 1). In hexadecane, the L1 and L2 biosurfactant had the highest EA and was comparable with the artificial surfactants. In diesel oil, the artificial surfactants had the highest activity compared to all three biosurfactants. The control tests had an expected activity close to zero in both hexadecane and diesel oil. The ES of the five compounds were calculated in hexadecane (Fig. 2) and diesel oil (Fig. 3). In hexadecane and diesel oil, all three biosurfactants were much more stable than the artificial surfactants.

5. Discussion
Overall, the L1, L2 and L3 had comparable EA and superior ES to the two artificial surfactants (SDS and Triton X-100) at same concentration (1g/L) in H20. Experimental error that could have affected our results include (1) impurities in extracted solid biosurfactant, including hexadecane and salts used in growth solution, (2) Soap contamination of glassware, increasing the observed emulsification activity, (3) Nonuniform vortexing when performed on several samples at once, leading to some to be stirred more than others before they are read in the spectrophotometer.

6. Further Work

Further work includes (1) Performing more emulsification property tests on Triton X100, SDS and control, (2) Determining if the difference in biosurfactant EA is statistically significant from the control and artificial surfactants (3) Varying pH, temperature and salinity, as biosurfactants have been cited as having a higher EA at these extremes, (4) Identifying the unknown bacterial strains using rRNA analysis (or similar assay) (5) Characterizing structure and size of the biosurfactants using GC-MS and SDS-PAGE, (6) Verifying the bacteria was secreting biosurfactant by measuring the change in surface tension over time, (7) Further purifying the biosurfactant extract from hexadecane and salt impurities, (8) Testing other artificial surfactants, such as cationic or nonionic and finally (9) Examining the possible enhancement of hydrocarbon-degrading bacteria by the addition of biosurfactant.

7. Conclusion
In this paper, we measured the emulsification properties of biosurfactants secreted from three biosurfactant-producing strains of bacteria native to the Yellow River Delta. Using a previously cited methods of measurement, we found that our three unknown biosurfactants (L1, L2, L3) had comparable emulsification activity and superior emulsification stability compared to the artificial surfactants tested (sodium dodecyl sulfate, Triton X-100).

8. Acknowledgements
This material is based on work supported by the National Science Foundation under Grant No. OISE-0229657. The results, discussions, and conclusions are those of the author and not of the National Science Foundation. The relevant research was conducted at Ocean University of China, Laoshan Campus under the supervision of Zhao Jian and Professor Zhenyu Wang. The author would like to thank Zhao Jian and Dr. Zhenyu Wang for their support, patience and teaching that resulted in this work. The author would also like to thank Dr. Hungtao Shen and Dr. Hayley Shen for organizing the 2008 Clarkson Marine Science and Engineering REU. The author would also like to thank the 2008 Qingdao interns for making this internship fun!

9. References
1. Xu, X., H. Lin, and Z. Fu, Probe into the method of regional ecological risk assessment--a case study of wetland in the Yellow River Delta in China. Journal of Environmental Management, 2004. 70(3): p. 253-262. Qi, S. and L. Fang, Environmental Degradation in the Yellow River Delta, Shandong Province, China. AMBIO: A Journal of the Human Environment, 2007. 36(7): p. 610-611. Singh, P. and S.S. Cameotra, Potential applications of microbial surfactants in biomedical sciences. Trends in Biotechnology, 2004. 22(3): p. 142-146. Rahman, P.K.S.M. and E. Gakpe, Production, Characterisation and Applications of Biosurfactants-Review. Biotechnology 2008. 7(2): p. 360-370. Menezes Bento, F., et al., Diversity of biosurfactant producing microorganisms isolated from soils contaminated with diesel oil. Microbiological Research, 2005. 160(3): p. 249-255. Zhang, G., et al., Biodegradation of crude oil by Pseudomonas aeruginosa in the presence of rhamnolipids. J Zhejiang Univ Sci B, 2005. 6(8): p. 725-30. Udemgba, C., Z. Jian, and W. Z.Y., The Biodegradation of Crude Oil by Autochthonous Degrading Bacteria from the Yellow River Delta 2007, College of Environmental Science and Engineering, Ocean University of China, Laoshan Campus, China. Wainwright, M., An Introduction to Environmental Biotechnology. 1st ed. 1999: Springer. 103.

2. 3. 4. 5. 6. 7.

8.

9. 10. 11. 12.

Surface Activity of Proteins: Chemical and Physicochemical Modifications. 1st ed, ed. S. Magdassi. 1996: CRC Press. 52. Pearce, K.N. and J.E. Kinsella, Emulsifying Properties of Proteins: Evaluation of a Turbidimetric Technique. J. Agric. Food Chem, 1978. 26(3): p. 716-723. Lee, S.-C., et al., Characterization of new biosurfactant produced by Klebsiella sp. Y6-1 isolated from waste soybean oil. Bioresource Technology, 2008. 99(7): p. 2288-2292. Pornsunthorntawee, O., et al., Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil. Bioresource Technology, 2008. 99(6): p. 1589-1595.

10. Figures

Figure 1. Emulsification activity of the three biosurfactants (L1, L2, L3), Triton X-100, SDS and with no surfactant (control). In hexadecane, their relative activities were 1.4713, 1.5975, 1.0465, 1.4020, 0.7750 and 0.1055, respectively. In diesel oil, their relative activities were 1.9258, 1.9553, 1.3947, 3.1700, 2.2070 and 0.1665, respectively.

Figure 2. Emulsification stability of the three biosurfactants (L1, L2, L3), Triton X-100 and SDS. (control not shown) over 60 minutes with error bars. Their relative stabilities were -0.002, -0.0008, -0.0009, -0.0076, -0.0076 and -0.00283 (control), respectively.

Figure 3. Emulsification stability of the three biosurfactants (L1, L2, L3), Triton X-100 and SDS (control not shown) over 60 minutes with error bars. Their relative stabilities were -0.0008, -0.0001, -0.0005, -0.0021, -0.0028 and -0.0022 (control), respectively.