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Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil
MPB-07637; No of Pages 6 Marine Pollution Bulletin xxx (2015) xxx–xxx
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Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil Boqun Liu a, Jinpeng Liu a,⁎, Meiting Ju a, Xiaojing Li b, Qilin Yu c a b c
College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China Agro-Environmental Protection Institute, Ministry of Agriculture, Tianjin 300191, PR China College of Life Sciences, Nankai University, Tianjin 300071, PR China
a r t i c l e
i n f o
Article history: Received 8 February 2016 Received in revised form 8 April 2016 Accepted 10 April 2016 Available online xxxx Keywords: Purification Biosurfactant Bacillus licheniformis Petroleum Soil
a b s t r a c t In our previous research, a petroleum degrading bacteria strain Bacillus licheniformis Y-1 was obtained in Dagang Oilfield which had the capability of producing biosurfactant. This biosurfactant was isolated and purified in this work. The biosurfactant produced by strain Y-1 had the capability to decrease the surface tension of water from 74.66 to 27.26 mN/m, with the critical micelle concentration (CMC) of 40 mg/L. The biosurfactant performed not only excellent stabilities against pH, temperature and salinity, but also great emulsifying activities to different kinds of oil, especially the crude oil. According to the results of FT-IR spectrum and 1H NMR spectrum detection, the surfactant was determined to be a cyclic lipopeptide. Furthermore, through the addition of surfactant, the effect of petroleum contaminated soil remediation by fungi got a significant improvement. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Petroleum contamination is one of the most severe environmental issues which attracts extensive attention. Large volumes of petroleum have been spilled in the environment owing to some inevitable oil spillages and accidents in the exploitation, transport and processing of oil (Ayotamuno et al., 2006). The massive spilled oil can cause extensive ecological damage to water, air and soil environments and have great impact on local economy (Bao et al., 2014). There are many toxic and harmful components found in the petroleum pollution, such as benzene, ethylbenzene, toluene, and xylenes (BETX) (Wang et al., 2012), which threaten human health. Synthetic surfactants are widely used in treating oil spills to disperse oil and accelerate its mineralization (Batista et al., 2006). By adding surfactants, the surface area of hydrophobic contaminants in soil or water was increased, resulting in the aqueous solubility and consequently the microbial degradation was increased (Karanth et al., 1999). However, synthetic surfactants used in environmental remediation are often toxic and harmful to the ecological function. Biosurfactants are surfactants produced by microorganism or plant which have similar properties to synthetic surfactants but are less toxic and more environmentally friendly. Numerous microorganisms were determined to have the capability to produce biosurfactants such ⁎ Corresponding author at: College of Environmental Science and Engineering, Nankai University, 94 Weijin Street, Tianjin 300071, PR China. E-mail address: [email protected] (J. Liu).
as Bacillus, Pseudomonas, Arthrobacter and Streptomyces (Dusane et al., 2011; Janek et al., 2010; Seghal Kiran et al., 2010). In most cases, biosurfactants are low-molecular mass compounds such as lipopeptide, glycolipid, and phospholipid or high-molecular mass lipoprotein, lipopolysaccharide, protein, polysaccharide and biopolymer complexes (Janek et al., 2010). The application of biosurfactants is not only bioremediation of petroleum contaminants but also reducing the heavy oil viscosity, enhancing oil recovery from wells, increasing flow though pipelines, cleaning oil storage tanks and stabilizing fuel water–oil emulsions (Batista et al., 2006). In our previous research, a petroleum-degrading bacterium Y-1 was obtained from petroleum contaminated soil in the Dagang Oilfield. The strain Y-1 was determined to produce biosurfactant at high temperature which has great emulsifying activity. The objectives of this work are to isolate and characterize the surfactant produced by strain Y-1.
2. Methods and materials 2.1. Materials The petroleum contaminated soil was made manually which contain uncontaminated soil obtained from Nankai University (Tianjin, China) and crude oil obtained from the oil pipeline of Dagang Oilfield (latitude: 38° 49′ N; longitude: 117° 31′ E). The fungi material used in remediation of petroleum contaminated soil was obtained through immobilizing fungi P-1 on straw. The fungi
http://dx.doi.org/10.1016/j.marpolbul.2016.04.025 0025-326X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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2.5. Determination of critical micelle concentration (CMC) With increasing concentration of surfactant, a sudden change in the decreasing rate of surface tension will happen at the CMC. By preparing different concentrations of surfactant solutions (0–80 mg/L) and measuring the changes of surface tension, CMC was determined from the inflection point of surface tension versus concentration (Cheng, 2013). 2.6. Determination of surfactant stability
Fig. 1. Growth rate and surface tension changes in cultivating of strain Y-1.
P-1 was identified as Pleurotus ostreatus and it was determined to have great petroleum hydrocarbon degradation ability.
2.2. Microorganism The strain Bacillus licheniformis Y-1 (GenBank accession number KP418813) used in this research was isolated from the area (latitude: 38° 49′ N; longitude: 117° 31′ E) near the pumping unit of the Dagang Oilfield in Tianjin, China. Characterization of strain Y-1 was described in our previous work (Boqun et al., 2016). In the cultivation of strain Y-1 in plate medium at 35 °C to 50 °C, a large amount of emplastic was discovered among the bacterial colonies and the amount increased with increasing temperature. It is proved that the emplastic produced by strain Y-1 exhibited a preferable emulsifying effect.
The stability of the surfactant was determined by surface tension changes under different conditions. The surfactant was dissolved in deionized water by the concentration of 500 mg/L. the stability against pH was assessed by varying the pH from 2 to 12; the stability against salinity was assessed by changing the concentration of NaCl (in the range of 0, 5, 10, 15, 20, 25, 30 g/L); and the temperature stability was studied by subjecting the solution to different temperatures (10, 20, 30, 40, 50, 60, 70, 80 °C) in an incubator for 120 min and then cooling to room temperature (Dubey et al., 2012). 2.7. Comparision with other common surfactant To evaluate the emulsifying activity of the surfactant, a comparison test was carried out. Several commercial surfactant such as Tween-80 and Rhamnolipid was selected to contrast with surfactant produced by strain Y-1. The emulsification index (E24) against the olive oil, diesel oil, crude oil and kerosene oil was determined. 5.0 mL of each oil was added to 5.0 mL of aqueous phase containing each surfactant. The mixture was agitated vigorously for 5 min on a vortex and kept undisturbed for 24 h at room temperature. The heights of the oil phase, the aqueous phase, and the emulsified layer were recorded. The E24% was determined by using the following equation (Chandankere et al., 2014): E24 % ¼ Height of the emulsifified layer=Total height of the liquid columnÞ 100
2.3. Changes of biomass and surface tension in cultivation The strain Y-1 was inoculated into the conical flask filled with T medium. Then the flask was placed on shaking table and rotated at 120 rpm to culture the strain at 35 °C. As the inoculation started, the strain was sampled every 4 h to determine the mycelia concentration therein. In other words, a UV spectrophotometer was adopted to measure the value of OD600 (Liu et al., 2014). After centrifuged for 20 min at 8000 r/min to remove the biomass, the surface tension of supernatant measurement was made with a JYW-200B automated tensiometer (Chengde Precision Instruments Co. Ltd., Hebei, China). The platinum ring was thoroughly rinsed with acetone and dried at 40 °C before each measurement.
2.8. Chemical characterization of the surfactant 2.8.1. FT-IR Fourier-transform infrared spectroscopy (FT-IR) analysis of the surfactant was performed with FTS 6000 FT-IR equipment (Bio-rad, USA).
2.4. Isolation and purification of surfactant After cultivated for 5 days, the fermentation broth was centrifuged twice at 8000 r/min for 20 min to remove the biomass. Crude surfactant was isolated by adding concentrated HCl to supernatant. A precipitate formed by pH 2 which could be collected, then washed by diluted hydrochloric acid twice. The precipitate was dissolved in dilute NaOH solution with a pH value of 7, followed by lyophilization. The crude surfactant was extracted with dichloromethane. The solvent was removed under reduced pressure to give a pale yellow solid. Further purification was achieved by recrystallization. The solid was dissolved in distilled water. This solution was filtered through Whatman no. 4 paper. The yellow solid was collected after lyophilization (Cooper et al., 1981).
Fig. 2. CMC of the surfactant produced by strain Y-1.
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
B. Liu et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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Fig. 4. Emulsification activities of surfactant produced by strain Y-1, Tween-80 and Rhamnolipid.
(Selb, Germany). About 10 mg of surfactant was loaded in a platinum pan and heated under a nitrogen atmosphere at 10 °C/min. The TG thermogram was obtained by plot-ting the weight percentage and heat flow against temperature in the range 25–900 °C. 2.9. Application in remediation of petroleum contaminated soil
Fig. 3. Influence of temperature (a), pH (b) and NaCl (c) on surfactant produced by strain Y-1.
For the further research on the surfactant produced by strain Y-1, the surfactant was applied in an experiment on remediation of petroleum contaminated soil. 10 mL (1%, m/v) surfactant aqueous solution was mixed with 200 g (dry weight) petroleum contaminated soil which contained 2.5% total petroleum hydrocarbon (TPH). Afterwards, 10 g (dry weight) fungi materials were inoculated into the soil. Meanwhile, a control group was established without adding surfactant. 5 mL deionized water was sprayed on the soil to keep moist. After 30 days remediation at 25 °C, the soil was sampled to determine petroleum hydrocarbon degradation rate. Extraction of TPH in soils was carried out using a modified method by Cai et al., 2010, (Cai et al., 2010). The low temperature dried soils were sieved through a 100 mesh, then weighed and extracted with dichloromethane (DCM) for 24 h using a Soxhlet apparatus. After reduced pressure distillation to remove the solvent, the petroleum hydrocarbons were weighted to calculated the TPH degradation rate. The hydrocarbons were dissolved in 5 mL of n-hexane and passed through column chromatography to separate the PAH. After reduced pressure distillation to remove the solvent, the PAH was weighted to calculated the PAH degradation rate. The measurements of 16 priority PAHs were performed on a gas chromatograph (GC, Agilent 7890 GC, US) equipped with Thermol Thermo Scientific TRA CETR-5MS GC Column (30 m 0.25 mm ID, 0.25 μm film thickness) with helium as carrier gas (1 mL/min). The following temperature program was used for PAH measurements: 70 °C for 1 min; and ramp at 10 °C/min to 260 °C for 4 min; then ramp at 5 °C/min to 300 °C and hold for 4 min (Wang et al., 2012). 3. Results and discussion
The purified product was diluted by KBr and dried under an infrared lamp to eliminate the influence of H2O. 2.8.2. 1H NMR The surfactant was dissolved in deuteroxide (D2O) and the 1H NMR spectra were recorded at 25 °C using a Bruker Avance 400 spectrometer operating. 2.8.3. Thermal gravimetric The thermal behavior of the surfactant was conducted in terms of thermal gravimetric (TG) on a NETZSCH TG209 thermal analysis system
3.1. Fermentation process monitoring In our previous work, the strain Y-1 was determined to have capability of producing surfactant at the temperature of over 35 °C. Therefore, we choose to monitor the growth of strain Y-1 at 35 °C. Fig. 1 shows that strain Y-1 entered the logarithmic phase at 8-28 h and the biomass in medium showed a rapid growth. In addition, the surface tension of supernate declined extremely since 16 h. It indicated that strain Y-1 began to produce surfactant with the multiplication in medium, resulting in the surface tension significantly declined. The lowest
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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Fig. 5. FT-IR spectrum (a), 1H NMR spectrum (b) and Thermogravimetric (TG) thermogram (c) of the surfactant produced by strain Y-1.
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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Fig. 6. GC–MS analysis of 16 PAHs in soil (NA, control group), TPH and PAH degradation rates.
surface tension was observed as 27.12 mN/m at 32 h, and it tended to stabilization in the remaining time. The surfactant reduced the surface tension from 68.88 mN/m to 27.12 mN/m, suggesting a higher surface activity compared with some published works (Cheng, 2013; Peypoux et al., 1994; Sajna et al., 2015). 3.2. Critical micelle concentration determination A very important characteristic of surfactant is the critical micelle concentration (CMC) which is defined as the surfactant concentration requisite to form micelle. When concentration of surfactant reaching the CMC, the surface tension remains relatively constant due to the interface saturation with the surfactant (Macdonald et al., 1981). Through applying surfactant in remediation of petroleum contaminated soil, the bioavailability of petroleum hydrocarbon gains a significantly increase (Bao et al., 2014; Chandankere et al., 2014; Sajna et al., 2015). However, at higher concentration, some surfactants may have inhibitory effect on the remediation as they exhibit antimicrobial activities (Whang et al., 2008). Therefore, it is significant to obtain a surfactant has not only low CMC but also high surface activity. As shown in Fig. 2, the CMC of the surfactant was 40 mg/L which was much lower than the reported SDS and Tween-80 (Jing, 2006), and the surface tension was reduced from 74.66 mN/m to 27.26 mN/m. These results indicated that the surfactant produced by strain Y-1 performed relatively well (Cheng, 2013; Pereira et al., 2013; Sajna et al., 2015). 3.3. Surfactant stability determination The stability in different conditions directly affects the applicability of surfactant. Three important factors were selected in this research, which were the temperature, the pH and the NaCl. From Fig. 3(a) we can see that, the surface tension had hardly changes from 10 °C to 80 °C. It is to say that the surface activity of surfactant produced by strain Y-1 was almost not influenced by the temperature of water. Such good thermostability has also been reported by Chandankere et al. (2014) and Dubey et al. (2012). Fig. 3(b) shows that the surface tension changed slightly when the pH value increased from 5 to 12. But when the pH value dropped below 4, the surface tension increased significantly which was due to the precipitation of surfactant. The similar result was also observed by Gong et al. (2009). The stability of surfactant produced by strain Y-1 performed well in the NaCl test
(Fig. 3(c)). Pretty high surface activity was observed when the concentration of NaCl was up to 30 g/L. 3.4. Comparision with other common surfactant Tween-80 and Rhamnolipid are both familiar surfactants which are widely applied in environmental modification research (Singh and Tripathi, 2013; Whang et al., 2008). So it would have much more representativeness to compare with these two surfactant. Fig. 4 reveals that the surfactant produced by strain Y-1 got the highest emulsification index in contrast with Tween-80 and Rhamnolipid. The emulsification index of surfactant produced by Y-1 was 91% for crude oil which was the maximum in this test, meanwhile, for other three kinds of oil it showed all more than 65%. The emulsification activities of Rhamnolipid were slightly higher than Tween-80. In experimenting we found that the crude oil was more easily to be emulsified than the other three kinds of oil while the kerosene oil was the most difficult, the emulsification index for crude oil of each three surfactants was beyond 80%, the familiar phenomenon also appeared in the research of Chandankere et al. (2014). 3.5. Chemical characterization of the surfactant As shown in Fig. 5(a), FT-IR spectrum reveals that there were several typical peaks of both peptide and aliphatic chain in the surfactant produced by strain Y-1. The strong peak at 3298 cm−1 shows the stretching of N\\H, furthermore the peak at 1658, 1581 and 1535 cm−1 shows the stretching of C_O and N\\H in peptide bonds respectively. At the fingerprint region of the spectra, a peak at 1094 cm−1 was observed which shows the vibration of C\\O\\C. The acetal structure in a sugar ring, confirming the existence of aliphatic chain. The peaks at 2954 cm−1 and 2927 cm−1 (with 2870 cm−1 symmetric stretching) belong to the stretching of the methyl and methylene. The peak at 3059 belongs to the hydroxyl which can be found in both aliphatic and peptide chains. The obvious peak at 1735 cm−1 shows the stretching of C_O in lactones. These results indicate that the surfactant produced by strain Y-1 is a cyclic lipopeptide. The 1H NMR spectrum of surfactant produced by strain Y-1 was shown in Fig. 5(b). There are three main regions correspond to resonance of amide protons (~ 8.42 ppm), α-carbon protons (3.57– 5.52 ppm), and side-chain protons (0.81–2.20 ppm). The peaks at 2.06
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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and 2.20 ppm indicate the presence of carbanyl group and peptide bond. The chemical shift at 1.15–1.30 ppm confirms the presence of a long aliphatic chain. The resonance at 3.57–3.93 ppm indicates the presence of hydroxyl group. The present 1H NMR spectrograms of surfactant are very similar to the standard surfactin (Sigma Co.) used in Wei's work (Wei and Chu, 1998), especially in the sections of 0.8–2.3 and 3.5– 5.5 ppm, signifying the similarity in molecular structure of surfactant produced by Y-1 with that of the surfactin standard. The only difference observed in 1H NMR spectra between them is in chemical shifts from the region correspondent to the amide protons, which is probably due to the different types of amino acid in each surfactant. The thermostability of biosurfactant is a critical property for various industrial applications (Chandankere et al., 2014). Some application in extreme conditions such as oil exploitation required the thermostability of surfactant particularly. Fig. 5(c) shows the thermal behavior of the surfactant produced by strain Y-1 which was assessed by TG and DTG. Three thermal degradation phases were observed in the whole process of heating up. A 1.80% weight loss of the surfactant was recorded at 80–200 °C in the first phase of degradation. The initial loss of weight probably due to moisture content, suggesting that the surfactant was not truly anhydrous. In the second phase, a 41.88% weight loss was observed at 200–630 °C. Finally, in the third phase of degradation, 8.46% weight loss was observed when the temperature rose up to 900 °C. A peak appeared in DTG curve at the temperature of 290.9 °C, which represent the maximum weight loss speed of the surfactant. The results obtained suggests that the surfactant produced by strain Y-1 has got a high thermostability and has a great application potential.
3.6. Application in remediation of petroleum contaminated soil After 30 d of remediation, the TPH and PAH degradation rate were measured by using weight differences method and the 16 PAHs concentration were measured by GC–MS analysis, as shows in Fig. 6. The TPH and PAH degradation rates of group without surfactant were 50.65 ± 2.99% and 40.68 ± 3.14% respectively. Compared with other studies, the fungi used in this study showed a higher level of degradation effect on TPH and PAH at the 30th day of remediation (Andersson et al., 2000; Li et al., 2016). Through adding surfactant produced by strain Y-1, the TPH and PAH degradation rates rose up to 54.00 ± 4.39% and 45.08 ± 6.63% respectively. It was because that the emulsification of surfactant made the petroleum hydrocarbon in the soil more easily to be utilized by microorganisms resulting in the improvement of the biodegradation effect of petroleum hydrocarbon in soil. In addition, as shown in Fig. 6, with the increase of the C number of the PAH, the microorganisms became more difficult to be degraded, which accords with the research results which employed other microbial technology for the remediation of petroleum-contaminated soil (Li et al., 2014; Wang et al., 2012). Significant improvement for the degradation of NP, ACE, FLN, PHE, PYR, BaA and BbF were observed, while slight improvement for the other 9 PAHs.
4. Conclusion The surfactant produced by petroleum hydrocarbon-degrading strain Y-1 was isolated and purified in this work. Through the FT-IR spectrum, 1H NMR spectrum detection, the surfactant was determined to be a cyclic lipopeptide. This surfactant possesses high surface activity which could reduce the surface tension of water from 74.66 mN/m to 27.26 mN/m at CMC of 40 mg/L and showed excellent emulsification activities against different kinds of oil. The stabilities under varying pH, temperature and salinity also provided the application in extreme conditions. Furthermore, through adding this surfactant, the effect of petroleum contaminated soil remediation by fungi got a significant improvement.
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Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil Boqun Liu a, Jinpeng Liu a,⁎, Meiting Ju a, Xiaojing Li b, Qilin Yu c a b c
College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China Agro-Environmental Protection Institute, Ministry of Agriculture, Tianjin 300191, PR China College of Life Sciences, Nankai University, Tianjin 300071, PR China
a r t i c l e
i n f o
Article history: Received 8 February 2016 Received in revised form 8 April 2016 Accepted 10 April 2016 Available online xxxx Keywords: Purification Biosurfactant Bacillus licheniformis Petroleum Soil
a b s t r a c t In our previous research, a petroleum degrading bacteria strain Bacillus licheniformis Y-1 was obtained in Dagang Oilfield which had the capability of producing biosurfactant. This biosurfactant was isolated and purified in this work. The biosurfactant produced by strain Y-1 had the capability to decrease the surface tension of water from 74.66 to 27.26 mN/m, with the critical micelle concentration (CMC) of 40 mg/L. The biosurfactant performed not only excellent stabilities against pH, temperature and salinity, but also great emulsifying activities to different kinds of oil, especially the crude oil. According to the results of FT-IR spectrum and 1H NMR spectrum detection, the surfactant was determined to be a cyclic lipopeptide. Furthermore, through the addition of surfactant, the effect of petroleum contaminated soil remediation by fungi got a significant improvement. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Petroleum contamination is one of the most severe environmental issues which attracts extensive attention. Large volumes of petroleum have been spilled in the environment owing to some inevitable oil spillages and accidents in the exploitation, transport and processing of oil (Ayotamuno et al., 2006). The massive spilled oil can cause extensive ecological damage to water, air and soil environments and have great impact on local economy (Bao et al., 2014). There are many toxic and harmful components found in the petroleum pollution, such as benzene, ethylbenzene, toluene, and xylenes (BETX) (Wang et al., 2012), which threaten human health. Synthetic surfactants are widely used in treating oil spills to disperse oil and accelerate its mineralization (Batista et al., 2006). By adding surfactants, the surface area of hydrophobic contaminants in soil or water was increased, resulting in the aqueous solubility and consequently the microbial degradation was increased (Karanth et al., 1999). However, synthetic surfactants used in environmental remediation are often toxic and harmful to the ecological function. Biosurfactants are surfactants produced by microorganism or plant which have similar properties to synthetic surfactants but are less toxic and more environmentally friendly. Numerous microorganisms were determined to have the capability to produce biosurfactants such ⁎ Corresponding author at: College of Environmental Science and Engineering, Nankai University, 94 Weijin Street, Tianjin 300071, PR China. E-mail address: [email protected] (J. Liu).
as Bacillus, Pseudomonas, Arthrobacter and Streptomyces (Dusane et al., 2011; Janek et al., 2010; Seghal Kiran et al., 2010). In most cases, biosurfactants are low-molecular mass compounds such as lipopeptide, glycolipid, and phospholipid or high-molecular mass lipoprotein, lipopolysaccharide, protein, polysaccharide and biopolymer complexes (Janek et al., 2010). The application of biosurfactants is not only bioremediation of petroleum contaminants but also reducing the heavy oil viscosity, enhancing oil recovery from wells, increasing flow though pipelines, cleaning oil storage tanks and stabilizing fuel water–oil emulsions (Batista et al., 2006). In our previous research, a petroleum-degrading bacterium Y-1 was obtained from petroleum contaminated soil in the Dagang Oilfield. The strain Y-1 was determined to produce biosurfactant at high temperature which has great emulsifying activity. The objectives of this work are to isolate and characterize the surfactant produced by strain Y-1.
2. Methods and materials 2.1. Materials The petroleum contaminated soil was made manually which contain uncontaminated soil obtained from Nankai University (Tianjin, China) and crude oil obtained from the oil pipeline of Dagang Oilfield (latitude: 38° 49′ N; longitude: 117° 31′ E). The fungi material used in remediation of petroleum contaminated soil was obtained through immobilizing fungi P-1 on straw. The fungi
http://dx.doi.org/10.1016/j.marpolbul.2016.04.025 0025-326X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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2.5. Determination of critical micelle concentration (CMC) With increasing concentration of surfactant, a sudden change in the decreasing rate of surface tension will happen at the CMC. By preparing different concentrations of surfactant solutions (0–80 mg/L) and measuring the changes of surface tension, CMC was determined from the inflection point of surface tension versus concentration (Cheng, 2013). 2.6. Determination of surfactant stability
Fig. 1. Growth rate and surface tension changes in cultivating of strain Y-1.
P-1 was identified as Pleurotus ostreatus and it was determined to have great petroleum hydrocarbon degradation ability.
2.2. Microorganism The strain Bacillus licheniformis Y-1 (GenBank accession number KP418813) used in this research was isolated from the area (latitude: 38° 49′ N; longitude: 117° 31′ E) near the pumping unit of the Dagang Oilfield in Tianjin, China. Characterization of strain Y-1 was described in our previous work (Boqun et al., 2016). In the cultivation of strain Y-1 in plate medium at 35 °C to 50 °C, a large amount of emplastic was discovered among the bacterial colonies and the amount increased with increasing temperature. It is proved that the emplastic produced by strain Y-1 exhibited a preferable emulsifying effect.
The stability of the surfactant was determined by surface tension changes under different conditions. The surfactant was dissolved in deionized water by the concentration of 500 mg/L. the stability against pH was assessed by varying the pH from 2 to 12; the stability against salinity was assessed by changing the concentration of NaCl (in the range of 0, 5, 10, 15, 20, 25, 30 g/L); and the temperature stability was studied by subjecting the solution to different temperatures (10, 20, 30, 40, 50, 60, 70, 80 °C) in an incubator for 120 min and then cooling to room temperature (Dubey et al., 2012). 2.7. Comparision with other common surfactant To evaluate the emulsifying activity of the surfactant, a comparison test was carried out. Several commercial surfactant such as Tween-80 and Rhamnolipid was selected to contrast with surfactant produced by strain Y-1. The emulsification index (E24) against the olive oil, diesel oil, crude oil and kerosene oil was determined. 5.0 mL of each oil was added to 5.0 mL of aqueous phase containing each surfactant. The mixture was agitated vigorously for 5 min on a vortex and kept undisturbed for 24 h at room temperature. The heights of the oil phase, the aqueous phase, and the emulsified layer were recorded. The E24% was determined by using the following equation (Chandankere et al., 2014): E24 % ¼ Height of the emulsifified layer=Total height of the liquid columnÞ 100
2.3. Changes of biomass and surface tension in cultivation The strain Y-1 was inoculated into the conical flask filled with T medium. Then the flask was placed on shaking table and rotated at 120 rpm to culture the strain at 35 °C. As the inoculation started, the strain was sampled every 4 h to determine the mycelia concentration therein. In other words, a UV spectrophotometer was adopted to measure the value of OD600 (Liu et al., 2014). After centrifuged for 20 min at 8000 r/min to remove the biomass, the surface tension of supernatant measurement was made with a JYW-200B automated tensiometer (Chengde Precision Instruments Co. Ltd., Hebei, China). The platinum ring was thoroughly rinsed with acetone and dried at 40 °C before each measurement.
2.8. Chemical characterization of the surfactant 2.8.1. FT-IR Fourier-transform infrared spectroscopy (FT-IR) analysis of the surfactant was performed with FTS 6000 FT-IR equipment (Bio-rad, USA).
2.4. Isolation and purification of surfactant After cultivated for 5 days, the fermentation broth was centrifuged twice at 8000 r/min for 20 min to remove the biomass. Crude surfactant was isolated by adding concentrated HCl to supernatant. A precipitate formed by pH 2 which could be collected, then washed by diluted hydrochloric acid twice. The precipitate was dissolved in dilute NaOH solution with a pH value of 7, followed by lyophilization. The crude surfactant was extracted with dichloromethane. The solvent was removed under reduced pressure to give a pale yellow solid. Further purification was achieved by recrystallization. The solid was dissolved in distilled water. This solution was filtered through Whatman no. 4 paper. The yellow solid was collected after lyophilization (Cooper et al., 1981).
Fig. 2. CMC of the surfactant produced by strain Y-1.
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
B. Liu et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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Fig. 4. Emulsification activities of surfactant produced by strain Y-1, Tween-80 and Rhamnolipid.
(Selb, Germany). About 10 mg of surfactant was loaded in a platinum pan and heated under a nitrogen atmosphere at 10 °C/min. The TG thermogram was obtained by plot-ting the weight percentage and heat flow against temperature in the range 25–900 °C. 2.9. Application in remediation of petroleum contaminated soil
Fig. 3. Influence of temperature (a), pH (b) and NaCl (c) on surfactant produced by strain Y-1.
For the further research on the surfactant produced by strain Y-1, the surfactant was applied in an experiment on remediation of petroleum contaminated soil. 10 mL (1%, m/v) surfactant aqueous solution was mixed with 200 g (dry weight) petroleum contaminated soil which contained 2.5% total petroleum hydrocarbon (TPH). Afterwards, 10 g (dry weight) fungi materials were inoculated into the soil. Meanwhile, a control group was established without adding surfactant. 5 mL deionized water was sprayed on the soil to keep moist. After 30 days remediation at 25 °C, the soil was sampled to determine petroleum hydrocarbon degradation rate. Extraction of TPH in soils was carried out using a modified method by Cai et al., 2010, (Cai et al., 2010). The low temperature dried soils were sieved through a 100 mesh, then weighed and extracted with dichloromethane (DCM) for 24 h using a Soxhlet apparatus. After reduced pressure distillation to remove the solvent, the petroleum hydrocarbons were weighted to calculated the TPH degradation rate. The hydrocarbons were dissolved in 5 mL of n-hexane and passed through column chromatography to separate the PAH. After reduced pressure distillation to remove the solvent, the PAH was weighted to calculated the PAH degradation rate. The measurements of 16 priority PAHs were performed on a gas chromatograph (GC, Agilent 7890 GC, US) equipped with Thermol Thermo Scientific TRA CETR-5MS GC Column (30 m 0.25 mm ID, 0.25 μm film thickness) with helium as carrier gas (1 mL/min). The following temperature program was used for PAH measurements: 70 °C for 1 min; and ramp at 10 °C/min to 260 °C for 4 min; then ramp at 5 °C/min to 300 °C and hold for 4 min (Wang et al., 2012). 3. Results and discussion
The purified product was diluted by KBr and dried under an infrared lamp to eliminate the influence of H2O. 2.8.2. 1H NMR The surfactant was dissolved in deuteroxide (D2O) and the 1H NMR spectra were recorded at 25 °C using a Bruker Avance 400 spectrometer operating. 2.8.3. Thermal gravimetric The thermal behavior of the surfactant was conducted in terms of thermal gravimetric (TG) on a NETZSCH TG209 thermal analysis system
3.1. Fermentation process monitoring In our previous work, the strain Y-1 was determined to have capability of producing surfactant at the temperature of over 35 °C. Therefore, we choose to monitor the growth of strain Y-1 at 35 °C. Fig. 1 shows that strain Y-1 entered the logarithmic phase at 8-28 h and the biomass in medium showed a rapid growth. In addition, the surface tension of supernate declined extremely since 16 h. It indicated that strain Y-1 began to produce surfactant with the multiplication in medium, resulting in the surface tension significantly declined. The lowest
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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Fig. 5. FT-IR spectrum (a), 1H NMR spectrum (b) and Thermogravimetric (TG) thermogram (c) of the surfactant produced by strain Y-1.
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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Fig. 6. GC–MS analysis of 16 PAHs in soil (NA, control group), TPH and PAH degradation rates.
surface tension was observed as 27.12 mN/m at 32 h, and it tended to stabilization in the remaining time. The surfactant reduced the surface tension from 68.88 mN/m to 27.12 mN/m, suggesting a higher surface activity compared with some published works (Cheng, 2013; Peypoux et al., 1994; Sajna et al., 2015). 3.2. Critical micelle concentration determination A very important characteristic of surfactant is the critical micelle concentration (CMC) which is defined as the surfactant concentration requisite to form micelle. When concentration of surfactant reaching the CMC, the surface tension remains relatively constant due to the interface saturation with the surfactant (Macdonald et al., 1981). Through applying surfactant in remediation of petroleum contaminated soil, the bioavailability of petroleum hydrocarbon gains a significantly increase (Bao et al., 2014; Chandankere et al., 2014; Sajna et al., 2015). However, at higher concentration, some surfactants may have inhibitory effect on the remediation as they exhibit antimicrobial activities (Whang et al., 2008). Therefore, it is significant to obtain a surfactant has not only low CMC but also high surface activity. As shown in Fig. 2, the CMC of the surfactant was 40 mg/L which was much lower than the reported SDS and Tween-80 (Jing, 2006), and the surface tension was reduced from 74.66 mN/m to 27.26 mN/m. These results indicated that the surfactant produced by strain Y-1 performed relatively well (Cheng, 2013; Pereira et al., 2013; Sajna et al., 2015). 3.3. Surfactant stability determination The stability in different conditions directly affects the applicability of surfactant. Three important factors were selected in this research, which were the temperature, the pH and the NaCl. From Fig. 3(a) we can see that, the surface tension had hardly changes from 10 °C to 80 °C. It is to say that the surface activity of surfactant produced by strain Y-1 was almost not influenced by the temperature of water. Such good thermostability has also been reported by Chandankere et al. (2014) and Dubey et al. (2012). Fig. 3(b) shows that the surface tension changed slightly when the pH value increased from 5 to 12. But when the pH value dropped below 4, the surface tension increased significantly which was due to the precipitation of surfactant. The similar result was also observed by Gong et al. (2009). The stability of surfactant produced by strain Y-1 performed well in the NaCl test
(Fig. 3(c)). Pretty high surface activity was observed when the concentration of NaCl was up to 30 g/L. 3.4. Comparision with other common surfactant Tween-80 and Rhamnolipid are both familiar surfactants which are widely applied in environmental modification research (Singh and Tripathi, 2013; Whang et al., 2008). So it would have much more representativeness to compare with these two surfactant. Fig. 4 reveals that the surfactant produced by strain Y-1 got the highest emulsification index in contrast with Tween-80 and Rhamnolipid. The emulsification index of surfactant produced by Y-1 was 91% for crude oil which was the maximum in this test, meanwhile, for other three kinds of oil it showed all more than 65%. The emulsification activities of Rhamnolipid were slightly higher than Tween-80. In experimenting we found that the crude oil was more easily to be emulsified than the other three kinds of oil while the kerosene oil was the most difficult, the emulsification index for crude oil of each three surfactants was beyond 80%, the familiar phenomenon also appeared in the research of Chandankere et al. (2014). 3.5. Chemical characterization of the surfactant As shown in Fig. 5(a), FT-IR spectrum reveals that there were several typical peaks of both peptide and aliphatic chain in the surfactant produced by strain Y-1. The strong peak at 3298 cm−1 shows the stretching of N\\H, furthermore the peak at 1658, 1581 and 1535 cm−1 shows the stretching of C_O and N\\H in peptide bonds respectively. At the fingerprint region of the spectra, a peak at 1094 cm−1 was observed which shows the vibration of C\\O\\C. The acetal structure in a sugar ring, confirming the existence of aliphatic chain. The peaks at 2954 cm−1 and 2927 cm−1 (with 2870 cm−1 symmetric stretching) belong to the stretching of the methyl and methylene. The peak at 3059 belongs to the hydroxyl which can be found in both aliphatic and peptide chains. The obvious peak at 1735 cm−1 shows the stretching of C_O in lactones. These results indicate that the surfactant produced by strain Y-1 is a cyclic lipopeptide. The 1H NMR spectrum of surfactant produced by strain Y-1 was shown in Fig. 5(b). There are three main regions correspond to resonance of amide protons (~ 8.42 ppm), α-carbon protons (3.57– 5.52 ppm), and side-chain protons (0.81–2.20 ppm). The peaks at 2.06
Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025
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and 2.20 ppm indicate the presence of carbanyl group and peptide bond. The chemical shift at 1.15–1.30 ppm confirms the presence of a long aliphatic chain. The resonance at 3.57–3.93 ppm indicates the presence of hydroxyl group. The present 1H NMR spectrograms of surfactant are very similar to the standard surfactin (Sigma Co.) used in Wei's work (Wei and Chu, 1998), especially in the sections of 0.8–2.3 and 3.5– 5.5 ppm, signifying the similarity in molecular structure of surfactant produced by Y-1 with that of the surfactin standard. The only difference observed in 1H NMR spectra between them is in chemical shifts from the region correspondent to the amide protons, which is probably due to the different types of amino acid in each surfactant. The thermostability of biosurfactant is a critical property for various industrial applications (Chandankere et al., 2014). Some application in extreme conditions such as oil exploitation required the thermostability of surfactant particularly. Fig. 5(c) shows the thermal behavior of the surfactant produced by strain Y-1 which was assessed by TG and DTG. Three thermal degradation phases were observed in the whole process of heating up. A 1.80% weight loss of the surfactant was recorded at 80–200 °C in the first phase of degradation. The initial loss of weight probably due to moisture content, suggesting that the surfactant was not truly anhydrous. In the second phase, a 41.88% weight loss was observed at 200–630 °C. Finally, in the third phase of degradation, 8.46% weight loss was observed when the temperature rose up to 900 °C. A peak appeared in DTG curve at the temperature of 290.9 °C, which represent the maximum weight loss speed of the surfactant. The results obtained suggests that the surfactant produced by strain Y-1 has got a high thermostability and has a great application potential.
3.6. Application in remediation of petroleum contaminated soil After 30 d of remediation, the TPH and PAH degradation rate were measured by using weight differences method and the 16 PAHs concentration were measured by GC–MS analysis, as shows in Fig. 6. The TPH and PAH degradation rates of group without surfactant were 50.65 ± 2.99% and 40.68 ± 3.14% respectively. Compared with other studies, the fungi used in this study showed a higher level of degradation effect on TPH and PAH at the 30th day of remediation (Andersson et al., 2000; Li et al., 2016). Through adding surfactant produced by strain Y-1, the TPH and PAH degradation rates rose up to 54.00 ± 4.39% and 45.08 ± 6.63% respectively. It was because that the emulsification of surfactant made the petroleum hydrocarbon in the soil more easily to be utilized by microorganisms resulting in the improvement of the biodegradation effect of petroleum hydrocarbon in soil. In addition, as shown in Fig. 6, with the increase of the C number of the PAH, the microorganisms became more difficult to be degraded, which accords with the research results which employed other microbial technology for the remediation of petroleum-contaminated soil (Li et al., 2014; Wang et al., 2012). Significant improvement for the degradation of NP, ACE, FLN, PHE, PYR, BaA and BbF were observed, while slight improvement for the other 9 PAHs.
4. Conclusion The surfactant produced by petroleum hydrocarbon-degrading strain Y-1 was isolated and purified in this work. Through the FT-IR spectrum, 1H NMR spectrum detection, the surfactant was determined to be a cyclic lipopeptide. This surfactant possesses high surface activity which could reduce the surface tension of water from 74.66 mN/m to 27.26 mN/m at CMC of 40 mg/L and showed excellent emulsification activities against different kinds of oil. The stabilities under varying pH, temperature and salinity also provided the application in extreme conditions. Furthermore, through adding this surfactant, the effect of petroleum contaminated soil remediation by fungi got a significant improvement.
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Please cite this article as: Liu, B., et al., Purification and characterization of biosurfactant produced by Bacillus licheniformis Y-1 and its application in remediation of petroleum contaminated soil..., Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2016.04.025