Marine Pollution Bulletin 101 (2015) 219–225
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Effect of rhamnolipid biosurfactant on solubilization of polycyclic aromatic hydrocarbons Shudong Li a,b, Yongrui Pi a,b, Mutai Bao a,b,⁎, Cong Zhang a,b, Dongwei Zhao a,b, Yiming Li a,b, Peiyan Sun c,d, Jinren Lu b a
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China College of Chemistry & Chemical Engineering, Ocean University of China, Qingdao 266100, China Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology, State Oceanic Administration, Qingdao 266033, China d North China Sea Environmental Monitoring Center of State Oceanic Administration, Qingdao 266033, China b c
a r t i c l e
i n f o
Article history: Received 16 July 2015 Received in revised form 29 September 2015 Accepted 30 September 2015 Available online 19 October 2015 Keywords: Rhamnolipid biosurfactant Polycyclic aromatic hydrocarbons (PAHs) Solubilization Naphthalene Phenanthrene Pyrene
a b s t r a c t Rhamnolipid biosurfactant-producing bacteria, Bacillus Lz-2, was isolated from oil polluted water collected from Dongying Shengli oilfield, China. The factors that influence PAH solubilization such as biosurfactant concentration, pH, ionic strength and temperature were discussed. The results showed that the solubilities of naphthalene, phenanthrene and pyrene increased linearly with the rise of rhamnolipid biosurfactant dose above the biosurfactant critical micelle concentration (CMC). Furthermore, the molar solubilization ratio (MSR) values decreased in the following order: naphthalene N phenanthrene N pyrene. However, the solubility percentage increased and followed the opposite order: pyrene N phenanthrene N naphthalene. The solubilities of PAHs in rhamnolipid biosurfactant solution increased with the rise of pH and ionic strength, and reached the maximum values under the conditions of pH 11 and NaCl concentration 8 g·L−1. The solubility of phenanthrene and pyrene increased with the rise of temperature. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Widespread use, improper disposal, incomplete combustion of fossil fuels and biomass, accidental spills and leaks of organic hydrocarbons like petroleum hydrocarbons, organic solvents, and polyaromatic hydrocarbons (PAHs) have resulted in long-term persistent sources of contamination of fossil oils, coal, tar deposits and groundwater, which becomes a major environmental issue because of their adverse effect on human health (Arias et al., 2010; Paria, 2008; Begoña et al., 2015). Polycyclic aromatic hydrocarbons (PAHs) are components of creosote produced during raw petroleum refining, coke production or wood preservation, and the distribution of PAH compounds in the marine sediments of rivers, estuaries, coastlines and sea beds has been commonly investigated (Boonyatumanond et al., 2006; Gong et al., 2014; Page et al., 1999; Pauzi Zakaria et al., 2001; da Silva and Bicego, 2010; Tolosa et al., 2004; Wang et al., 2011). The structure of PAHs is composed of two or more benzene rings covalently bonded together. With the number of rings in the molecular structure increases, water solubility decreases, and hence makes it more difficult to degrade. The toxicity of PAHs including possible mutagenic and carcinogenic ⁎ Corresponding author at: Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China. E-mail address: [email protected] (M. Bao).
http://dx.doi.org/10.1016/j.marpolbul.2015.09.059 0025-326X/© 2015 Elsevier Ltd. All rights reserved.
effects may exert potential health risk to urban residents (Colombo et al., 2006; Al-Saleh et al., 2013), as well as it can be bio-accumulated through the food chain and the exposure of humans to PAHs may enhance the risk of cancer and other adverse health effects (Gu et al., 2013; Wan et al., 2007). PAHs also have a strong inhibitory effect on microbial growth (Calder and Lader, 1976) and can destroy the biological membrane and damage DNA, causing genetic information of the cells to be mutated (Kim et al., 2007; Jarvis et al., 2013). As PAHs are so harmful to the environment and human health, it is important to find effective degradation methods. Surfactants are amphiphilic compounds that can reduce the free energy of the system by replacing the bulk molecules of higher energy at the interface. The term surface-active agent or “surfactant” represents a heterogeneous and long-chain molecule containing both hydrophilic (head) and hydrophobic (tail) moieties (Paria, 2008). They contain a hydrophobic portion with little affinity for the bulk medium and a hydrophilic group attracted to the bulk medium. Consequently, surfactants are used to lower surface tensions and increase solubility. They are used for their detergency power, wetting ability and foaming capacity in petroleum industry (Al-Sabagh et al., 2003), mineral flotation (Asplin et al., 1998; Koopal et al., 1999) and pharmaceutical industries (Madunić-Čačić et al., 2008; Christiansen et al., 2011; Nogueira et al., 2011). But the introduction of surfactants into the environment can lead to contamination concerns. Consequently, the toxicity of the
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surfactant and its potential degradation products needs to be carefully considered prior to the selection of surfactant (Edwards et al., 2003). Biosurfactants are mainly produced by bacteria or yeast, and also available from plants, animals, including human (Paria, 2008). Most microbial surfactants are complex molecules, comprising different structures that include lipopeptides, glycolipids, polysaccharide protein complexes, fatty acids and phospholipids (Thavasi et al., 2008). In the past few decades, biosurfactants have gained more attention and more potentially effective over synthetic surfactants because of their advantages such as high specificity biodegradability and biocompatibility (Torres et al., 2011), low toxicity, ecological acceptability and ability to be produced from renewable and cheaper substrates (Desai and Banat, 1997; Nitschke and Pastore, 2004; Paria, 2008). Therefore, there have more environmental applications in recent years about biosurfactants. Most of the studies focused on their application in the remediation of wastewaters; soils contaminated by heavy metals and hydrophobic organic compounds, such as petroleum hydrocarbons and pesticides (Sponza and Gök, 2010; Zeftawy et al., 2011; Thavasi et al., 2011; Shin et al., 2006; Mulligan and Wang, 2006; Wang and Mulligan, 2009; Cao et al., 2013). A recent study done by Zhou (Zhou et al., 2011) about saponin, a plant-derived non-ionic biosurfactant, described its application in enhancing solubilization of PAHs; it showed that the solubility of PAHs was highly dependent of pH and ionic strength of the solution; the molar solubilization ratio (MSR) of saponin for phenanthrene was about 3–6 times of those synthetic non-ionic surfactants, and decreased about 70% with the increase of solution pH from 4.0 to 8.0, but increased linearly when NaCl concentration increased from 0.01 to 1.0 M; saponin is more effective in enhancing PAH solubilization than synthetic non-ionic surfactants and has potential application in removing organic pollutants from contaminated soils. In this research, we analyzed the structure of a biosurfactant that was produced from oil degradation bacteria Bacillus Lz-2. The corresponding influences of a number of environmental factors (biosurfactant concentrations, pH, ionic strength and temperature) were investigated. The results improve the understanding and prediction of the enhanced solubilization of rhamnolipid biosurfactant for PAHs and provide valuable information for the application of biosurfactants in the remediation of organic contaminated water. The main components of the oil are alkanes, cyclanes and aromatic hydrocarbons. Aromatic hydrocarbons are difficult to be degradated. Rhamnolipid biosurfactant can enhance the solubilization of PAHs in experiment (Zhao et al., 2015), so it can work on other simple components in the oil. Rhamnolipid biosurfactant enhances certain solubilization on oil and achieves certain auxiliary degradation effect. So it can be applied to the marine oil spill.
was 99% similar to Bacillus based on its 16S rRNA gene sequence (1342 bp) and was named Bacillus Lz-2 (GenBank accession number KC256826). The critical micellar concentration (CMC) of this biosurfactant is 240 mg·L−1. The mineral salt medium (MSM) with supplement of sodium citrate 30 g·L−1, peptone 5 g·L−1, NaCl 5 g·L−1; pH = 7, T = 37 °C was used for bacterial growth. For inoculation, the strain was incubated in autoclaved MSM for a week on DHZ-D constant temperature shaker (Taicang Lab-Line Instruments, China) at 150 rpm. After centrifuged at 12,000 r·min− 1 at 4 °C for 20 min, the supernatant was adjusted to pH 2.0 with 1.0 M sulfuric acid, prior to biosurfactant extraction using equal volume of ethyl acetate. The organic phase was separated and the solvent was evaporated to concentrate the biosurfactant. The biosurfactant was then dried using a rotary evaporator. The dried product was washed with absolute ethanol three times completely to remove residual pigments. The IR spectra were recorded on Bruker IFS66 V FT-IR spectrometer (KBr and polyethylene pellets). The FT-Raman and SERS spectra were obtained on a Bruker IFS66V NIR-FT instrument equipped with a FRA 106 Raman module. A Nd/YAG laser at 1064 nm with an output of 300 mW was used for excitation. The detector was a Ge-diode cooled to liquid nitrogen temperature. One thousand scans were accumulated with a total registration time of about 30 min. The spectral resolution was 6 cm−1. HPLC–MS characterization of the biosurfactant was achieved using the quadrupole ion-trap mass spectrometer (1290–6430, Agilent, USA). The scanning type was positive/negative ion scanning with scan range m/z 200–1500. The fragmentor and capillary voltage were set to 110 V and 4.5 kV, and the drying gas temperature was 350 °C with the flow 9 L·min−1. HPLC–MS separation of the rhamnolipid mixtures utilized a reverse phase ZOBARX SB C18 column (5 μm C18, 100 × 2.1 mm, Agilent) and a binary gradient mobile phase comprising HPLC grade H2O as mobile phase A and methanol as mobile phase B. Initially, the proportions were 30% A and 70% B. The flow rate of 0.2 ml min−1 and an injection volume of 1 μL were used throughout.
2. Methods and materials
2.4. PAH detection and quantification
2.1. Chemicals
Detection and quantification of different PAHs (naphthalene, phenanthrene and pyrene) were carried out using Ultraviolet Spectrophotometry (UV2450, Shimadzu).
Naphthalene, phenanthrene and pyrene (purity N98%) were obtained from Aldrich Chemical Company. They were chosen as the three PAH probes because they are petroleum-, coal- and chemical industryrelated organic contaminants that are frequently found in surface and subsurface environments. Dichloromethane and sodium hydroxide were purchased from the Chinese Medicine Group Chemical Reagent Co., LTD. Sulfuric acid was purchased from Tianjin Yao Hua Chemical Reagent Co., LTD. Sodium chloride was used to investigate the effect of ionic strength on PAH solubilization. HCl and NaOH were employed in studying the effect of solution pH on PAH solubilization. 2.2. Purification and identification of the biosurfactant The biosurfactant used in this study was a rhamnolipid surfactant obtained from a bacterial strain, which had been isolated from oil polluted water collected from Dongying, China. The isolated bacteria
2.3. Preparation of standard solution 0.2 g of each PAH compound was dissolved in 100 mL of dichloromethane to prepare a stock solution of 2 g·L−1. The solution was then stored at −10 °C. The PAHs were quantified using 7 levels of external standards obtained by serial dilutions of stock solutions at a concentration range 2–14 mg·L−1 of naphthalene, 0.4–2.8 mg·L− 1 of phenanthrene, 0.2–2.6 mg·L−1 of pyrene.
2.5. Solubilization experiments All experiments were carried out in 100 mL glass conical flasks with Teflon-lined screw caps. The effects of biosurfactants on the solubilization behaviors of naphthalene, phenanthrene and pyrene were examined. For the solubilization experiment, excess PAHs were added to the conical flask and 25 mL background solution. The background solution was comprised of appropriate biosurfactant with 0.01 M NaNO3, which was a biocide system. The pH was adjusted to 7 at a constant ion concentration. The conical flasks were placed on a reciprocal shaker at 30 °C, 100 rpm for 48 h to reach solubilization equilibrium. Preliminary experiments showed that 48 h were enough for the solubilization of phenanthrene to reach equilibrium, and phenanthrene degradation or adsorption by the tubes was negligible. The suspensions
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were then centrifuged at 10,000 rpm for 25 min. Phenanthrenes in the aqueous phase were extracted with dichloromethane, and their concentrations were analyzed by Ultraviolet Spectrophotometry (UV2450, Shimadzu). The UV wavelengths of naphthalene, phenanthrene and pyrene were 228, 254 and 274 nm. All the tests were conducted in triplicate. 2.6. Effect of the biosurfactant concentrations, ionic strength, pH and temperature on the solubilization of naphthalene, phenanthrene and pyrene To investigate the effect of biosurfactant concentrations on the solubilization of naphthalene, phenanthrene, and pyrene, the solubility tests were done using different biosurfactant concentrations (0, 0.112, 0.224, 0.336, 0.448, 0.560 g·L−1). The initial pH value was 7. Then tests were performed in the same manner as the batch solubilization experiments described above. To investigate the effect of ionic strength, the solubility tests were conducted in the presence of biosurfactant and different concentrations of NaCl, which was added at different concentrations (1, 2, 4, 6, 8 and 10 g·L−1) with initial pH = 7. Then the tests were performed in the same manner as the batch solubilization experiments described above. The nature of biosurfactant can vary at different pH. So the solubilization of naphthalene, phenanthrene and pyrene under different pH conditions were studied. Prior to the test, pH of background solution was adjusted to 3, 5, 7, 9, 11 and 13 with the appropriate concentration of HCl and NaOH respectively, the biosurfactant concentration was kept at 0.56 g·L−1. Again, the tests were performed in the same manner as the batch solubilization experiments described above. To investigate the effect of the temperature, reciprocal shakers were respectively set at 25, 30, and 35 °C. The initial concentration for each biosurfactant was 0.56 g·L− 1 with an initial pH of 7. Then the tests were conducted in the same manner as the batch solubilization experiments described above. 3. Results and discussion 3.1. The structure of biosurfactant 3.1.1. FT-IR spectrum There are several literature reports on isolation and biosurfactant production by different species of bacteria. The molecular composition of biosurfactant produced by Lz-2 was evaluated using FT-IR (Fig. 1). The characteristic band at 3386 cm−1 indicates the presence of –OH bonds. Absorption around 2942 cm−1, 2929 cm−1 and 2853 cm−1 is assigned to the symmetric stretch –CH, –CH2 and –CH3 groups of
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aliphatic chains, and the absorption around 1259 cm−1 and 1371 cm−1 is assigned to the deformation vibrations of –CH. The absorption peak located at 1731 cm−1 indicates the presence of ester carbonyl groups (C=O bond in –COOH). The absorption peak around 1055 cm−1 indicates the presence of polysaccharide or polysaccharide-like substances. The absorption peak at 660 cm−1 indicates the presence of –CH2 group. The above information from the respective wave numbers was confirmed with the glycolipid nature of the biosurfactant. In a previous publication (Aparna et al., 2012), the structure of a rhamnolipid, produced by Pseudomonas sp. 2B was similar to the structure in our research. 3.1.2. HPLC–MS characterization of the rhamnolipid To identify the chemical structure of the product, an aliquot of the purified rhamnolipids produced by Lz-2 was analyzed by HPLC–MS. This analysis revealed 13 homologs with the pseudomolecular ions being between m/z 451 and 809 (Fig. 2). These results were in accordance with the previous studies on rhamnolipids (Manresa et al., 2003; Mulligan et al., 2005; Wray et al., 2007; Paria, 2008; Ibrahim et al., 2010). The presence of rhamnolipid in the crude extract was detected by HPLC–ESI+ MS and HPLC–ESI −MS (Table 1). The main components appeared as sodiated molecular ion clusters in the ESI + MS and [M + Na]+. In the ESI+MS, datum were observed at m/z 453.4 (Rha-C8–C8), 475.3 (Rha-C8–C10, Rha-C10–C8), 679.6 (Rha–Rha-C10– C10 ) and 701.5 (Rha–Rha-C10 –C 12, Rha–Rha-C 12 –C10 ), where Rha—indicates a rhamnose moiety. More detailed information about the structures of the rhamnolipids was derived by ESI −MS, which confirmed the presence of Rha-C8–C8, Rha-C10–C8, Rha-C10– C10, Rha-C 12 –C10, Rha–Rha-C10− C12, Rha–Rha-C12 –C 12. According to the intensities of the fragment ions, the shorter fatty acid is found preferentially directly linked to the rhamnose moiety in LZ-2. The combined data from fragmentation in HPLC–ESI+MS and those from HPLC–ESI −MS allowed the rhamnolipid content of each fraction to be rapidly assessed and showed this combination of techniques to be a very efficient tool for the detailed analysis of complex rhamnolipid mixtures after a simple separation. Therefore, applying LC–MS to rhamnolipid mixtures is an efficient technique because it enables compounds to be identified and chromatographically unresolved pairs of congeners to be quantified. 3.2. The standard curve of the PAHs The standard curves of naphthalene, phenanthrene and pyrene in dichloromethane were measured by UV method. The results were showed in Fig. 3. The standard curves of naphthalene, phenanthrene and pyrene in dichloromethane were Y = 0.108 + 0.05636 ∗ X, R2 = 0.9795; Y = −0.00743 + 0.34723 ∗ X, R2 = 0.9909; Y = −0.02418 + 0.24054 ∗ X, R2 = 0.9941. 3.3. Effect of the biosurfactant concentrations on the solubilization of PAHs
Fig. 1. The FT-IR spectrum of the crude biosurfactant.
Various biosurfactant concentrations were applied to evaluate the performance of biosurfactant in dissolved PAH aqueous phase. As Fig. 4 shows, without the biosurfactant, the concentrations of naphthalene, phenanthrene, and pyrene in aqueous phase were 5.18, 0.9 and 0.28 mg·L− 1. However, in the presence of 0.56 g·L− 1 biosurfactant, they reached 11.91, 2.07 and 1.32 mg·L−1, which corresponds to increase in solubility increased by 129%, 131%, and 372%, respectively. Therefore, the biosurfactant concentration is a critical factor for the solubilization of PAHs. This shows that the presence of biosurfactant has a significant impact on solubilization of PAHs. Upon further experiments, it was observed that PAH solubilization enhanced much more with the increase of biosurfactant concentration (As seen in Fig. 4). When the concentration is above the CMC, hydrophobic pollutants can readily partition into the hydrophobic core at the center of a micelle, thus increasing PAH aqueous concentration through micelle solubilization and promoting solubilization of PAHs.
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Fig. 2. The ESI–MS of the rhamnolipid fractions of purified sample of LZ-2. a) Negative-ion. b) Positive-ion.
The results of the percentage increase still illustrated with the increase of PAH molecular weight, the solubility of the PAHs decreased, but the solubility of percentage increased. Molar solubilization ratio (MSR) (Edwards et al., 1991) in Eq. (1) was described as the ability of PAHs by the biosurfactant. MSR ¼ SPAH; mic −SPAH; cmc =ðC surf −CMCÞ
ð1Þ
Here, Csurf is the concentration of biosurfactant above CMC, SPAH, cmc is the solubility of PAHs at CMC, SPAH, mic is the solubility of PAHs at Csurf. The MSR values were shown in Table 2. Naphthalene has the highest MSR value among the three PAHs, while pyrene has the lowest. In the presence of biosurfactant, PAH solubility decreases as the number of
Table 1 Identification and characterization of rhamnolipid from LZ-2 using HPLC–MS. Major compounds by ESI+MS
Major compounds by ESI−MS
[M + Na] m/z
Substance
[M − H]− m/z
Substance
453.40 475.30 679.60 701.50
Rha-(C8–C8) Rha-(C8–C10) Rha–Rha-(C10–C10) Rha–Rha-(C10–C12)
451.30 487.30 520.30 563.30 583.30 677.50 713.50 740.50
Rha-(C8–C8) Rha-(C8–C10) Rha-(C10–C10) Rha-(C12–C10) Rha-(C12–C12) Rha–Rha-(C10–C10) Rha–Rha-(C10–C12) Rha–Rha-(C12–C12)
+
rings in PAH structure increases. This explains the significance of molecular structure on PAH solubility. 3.4. Effect of ionic strength on the solubilization of PAHs It is well known that ionic strength can affect the micelle aggregation number and CMC values of ionic surfactants. Hence the solubilization capabilities of ionic surfactants for PAHs were examined by changing NaCl concentrations (from 1 to 10 g·L−1) at pH = 7; the results are shown in Fig. 5. As NaCl concentration increases, the solubility of PAHs increases linearly. When the concentration of NaCl jumps from 0 to 2 g·L−1, the solubility of PAHs increases significantly. For the ionic surfactant, adding NaCl electrolyte, CMC of the ionic surfactant is decreased and the logarithm of CMC had a linear relationship with the counter ion concentration values in the system. However, the amplitude of the CMC reducing was decreased as the concentration of NaCl increased beyond the value of 8 g·L− 1. When the concentration of NaCl is 8 g·L−1, the solubility of PAHs reached the maximum (Fig. 5), while the solubility of naphthalene, phenanthrene and pyrene were 2.27, 1.57, and 0.98 mg·L−1 respectively. Due to the presence of electrolyte, the electric double layer of the micelle was compressed, reducing the mutual repulsion between the ionic head surfactant and resulting the surfactant ions into the micelles. The aggregation number greatly increased and the number of the PAHs in the micelle increased too, which was similar with the findings of Zhou et al. (2011). For the non-ionic surfactant, however, the addition of electrolyte at low level does not have obvious effect on the enhanced solubilization of nonionic surfactant for PAHs, but a small change occurs at high electrolyte
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Fig. 4. Different concentrations of surfactant solution on naphthalene, phenanthrene and pyrene solubilization.
(RL) formed either lamella-like structures or lipid aggregates; and for pH N 6.8, the micelles were formed when the rhamnose moiety is negatively charged. Using a transmission electron microscopy, Champion (Champion et al., 1995) observed the RL structure decreasing in size as the pH increased from 5.5 to 8.0. This is due to repulsion between the more negatively charged head groups effectively creating a larger head diameter, and then causing changes in the morphology from lamellar to vesicles to micelles. Based on our results, coupled with the results of Ishigami (Ishigami et al., 1987) and Champion (Champion et al., 1995), we hypothesize that the RL solubility for PAHs increase as the morphology changes when the pH increases from 3 to 11. The results indicated that the solution pH had significant effect on the solubilization capabilities of biosurfactant for PAHs. 3.6. Effect of temperature on the solubilization of PAHs
Fig. 3. The standard curve of a) naphthalene, b) phenanthrene and c) pyrene in dichloromethane.
concentrations due to the “salting out” of the non-ionic surfactants (Paria, 2008). 3.5. Effect of pH on the solubilization of PAHs In general, pH has an obvious effect on the solubilization of ionic surfactants for PAHs. The biosurfactant molecule used in this study is composed of glucuronic acid as part of its head group, and thus the pH has a potential effect on the species of biosurfactant molecule and enhanced solubilization of PAHs. The effect of pH on the solubilization of PAHs in biosurfactant solution was investigated with changing the pH from 3 to 13. With the pH increasing, the solubility of PAHs increased and reached the maximum value of pH 11, as shown in Fig. 6. The corresponding maximum solubility of naphthalene, phenanthrene and pyrene were 4.47, 2.43 and 1.6 mg·L− 1. The micelle structure was changed from flake to vesicular, and finally into micelles, indicating a gradual decrease in its structure. Using fluorescence microscopy, Ishigami (Ishigami et al., 1987) reported that rhamnolipid (RL) form liposome-like vesicles at pH ≤ 6; for pH values of 6–6.6, the rhamnolipid
Temperature has a significant effect on solubilization of organic compounds in the presence of surfactant. For both ionic and nonionic surfactants, an increase in temperature generally results in an increase in the extent of solubilization for both polar solubilization and nonpolar solubilization (Paria, 2008). The effect of temperature on the solubilization of PAHs in the presence of biosurfactant, may be influenced by the characters of the biosurfactant or PAHs. The effect was attributed to (a) the changes in the aqueous phase solubility of the organic compound, and (b) changes in the surfactant micelles with temperature (Yeom et al., 1995). For the ionic biosurfactant, temperature has little effect. However temperature has great influence on the solubility of PAHs. Micelles formed in these aqueous block copolymer systems as the result of warming. Hence, as the temperature of the aqueous system rises, the onset of micelles should provide the mechanism whereby the apparent aqueous solubility of a hydrophobic solute rises dramatically. The impact of temperature on the solubility for naphthalene, phenanthrene and pyrene was shown in Fig. 7. With the temperature increasing, the solubility of phenanthrene and pyrene was increasing, possibly Table 2 The solubility of the PAHs by the biosurfactant. PAHs
Naphthalene Phenanthrene Pyrene
MSR
The solubility of the PAHs/mg·L−1 Pure water
CMC
5.18 0.9 0.28
6.1 1.01 0.65
7.44 2.83 1.34
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Fig. 5. Different ionic strengths on naphthalene phenanthrene and pyrene solubilization by surfactant solution.
due to an increase in the number of micelles. When the temperature goes above 30 °C, the solubility of naphthalene decreased which may be caused by the fact that naphthalene volatilized faster with increasing temperature, therefore the solubility of naphthalene in aqueous decreases. With the increase of the ring, the hydrophobic of PAHs increased, the volatile of the PAHs reduced, while the number of micelles increased, thus the solubility of phenanthrene and pyrene increased. The increasing apparent solubility of phenanthrene and pyrene over an extended temperature range suggested a gradual increased in the micellar composition in solution as a result of increasing temperature.
Fig. 7. Different temperatures on naphthalene phenanthrene and pyrene solubilization by surfactant solution.
and 1.6 mg·L−1 when pH was at 11; while the solubility of naphthalene phenanthrene and pyrene were 2.27, 1.57 and 0.98 mg·L−1 when the concentration of NaCl was 8 g·L− 1. The solubility of phenanthrene and pyrene increased with temperature, but the solubility of naphthalene first increased with increasing temperature but then decreased, reaching its maximum solubility at 30 °C. This solubilization switching should be of significant interest to those interested in the technical uses of block co-polymer surfactants. Furthermore, the results from this work also provide a useful assessment tool for rapid selection of biosurfactant conditions for their effectiveness on removing petroleum hydrocarbons from polluted water.
4. Conclusion The results obtained in this study demonstrated the sensitivity of PAH solubilization in micelles of biosurfactant to changes in solution phase pH, temperature and the ionic strength. In particular, the solubility of naphthalene, phenanthrene and pyrene increased linearly with rhamnolipid biosurfactant dose above the biosurfactant CMC. The solubility of PAHs in rhamnolipid biosurfactant solution increased with pH and ionic strength, and reached the maximum value under the pH value of 11 or the NaCl concentration value of 8 g·L− 1. The solubility of naphthalene, phenanthrene and pyrene were 4.47, 2.43
Acknowledgments This research was founded by the “National Natural Science Foundation of China” (41376084); the Major Projects of National High Technology Research and Development Program 863 (2013AA064401); the Public Science and Technology Research Funds Projects of Ocean (201205012); the “Program for Innovative Research Team in University” (IRT1289); and the Open Foundation of Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology of SOA (201402). This is MCTL Contribution No. 104.
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Fig. 6. Different pH on naphthalene, phenanthrene and pyrene solubilization, by surfactant solution.
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Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Effect of rhamnolipid biosurfactant on solubilization of polycyclic aromatic hydrocarbons Shudong Li a,b, Yongrui Pi a,b, Mutai Bao a,b,⁎, Cong Zhang a,b, Dongwei Zhao a,b, Yiming Li a,b, Peiyan Sun c,d, Jinren Lu b a
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China College of Chemistry & Chemical Engineering, Ocean University of China, Qingdao 266100, China Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology, State Oceanic Administration, Qingdao 266033, China d North China Sea Environmental Monitoring Center of State Oceanic Administration, Qingdao 266033, China b c
a r t i c l e
i n f o
Article history: Received 16 July 2015 Received in revised form 29 September 2015 Accepted 30 September 2015 Available online 19 October 2015 Keywords: Rhamnolipid biosurfactant Polycyclic aromatic hydrocarbons (PAHs) Solubilization Naphthalene Phenanthrene Pyrene
a b s t r a c t Rhamnolipid biosurfactant-producing bacteria, Bacillus Lz-2, was isolated from oil polluted water collected from Dongying Shengli oilfield, China. The factors that influence PAH solubilization such as biosurfactant concentration, pH, ionic strength and temperature were discussed. The results showed that the solubilities of naphthalene, phenanthrene and pyrene increased linearly with the rise of rhamnolipid biosurfactant dose above the biosurfactant critical micelle concentration (CMC). Furthermore, the molar solubilization ratio (MSR) values decreased in the following order: naphthalene N phenanthrene N pyrene. However, the solubility percentage increased and followed the opposite order: pyrene N phenanthrene N naphthalene. The solubilities of PAHs in rhamnolipid biosurfactant solution increased with the rise of pH and ionic strength, and reached the maximum values under the conditions of pH 11 and NaCl concentration 8 g·L−1. The solubility of phenanthrene and pyrene increased with the rise of temperature. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Widespread use, improper disposal, incomplete combustion of fossil fuels and biomass, accidental spills and leaks of organic hydrocarbons like petroleum hydrocarbons, organic solvents, and polyaromatic hydrocarbons (PAHs) have resulted in long-term persistent sources of contamination of fossil oils, coal, tar deposits and groundwater, which becomes a major environmental issue because of their adverse effect on human health (Arias et al., 2010; Paria, 2008; Begoña et al., 2015). Polycyclic aromatic hydrocarbons (PAHs) are components of creosote produced during raw petroleum refining, coke production or wood preservation, and the distribution of PAH compounds in the marine sediments of rivers, estuaries, coastlines and sea beds has been commonly investigated (Boonyatumanond et al., 2006; Gong et al., 2014; Page et al., 1999; Pauzi Zakaria et al., 2001; da Silva and Bicego, 2010; Tolosa et al., 2004; Wang et al., 2011). The structure of PAHs is composed of two or more benzene rings covalently bonded together. With the number of rings in the molecular structure increases, water solubility decreases, and hence makes it more difficult to degrade. The toxicity of PAHs including possible mutagenic and carcinogenic ⁎ Corresponding author at: Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China. E-mail address: [email protected] (M. Bao).
http://dx.doi.org/10.1016/j.marpolbul.2015.09.059 0025-326X/© 2015 Elsevier Ltd. All rights reserved.
effects may exert potential health risk to urban residents (Colombo et al., 2006; Al-Saleh et al., 2013), as well as it can be bio-accumulated through the food chain and the exposure of humans to PAHs may enhance the risk of cancer and other adverse health effects (Gu et al., 2013; Wan et al., 2007). PAHs also have a strong inhibitory effect on microbial growth (Calder and Lader, 1976) and can destroy the biological membrane and damage DNA, causing genetic information of the cells to be mutated (Kim et al., 2007; Jarvis et al., 2013). As PAHs are so harmful to the environment and human health, it is important to find effective degradation methods. Surfactants are amphiphilic compounds that can reduce the free energy of the system by replacing the bulk molecules of higher energy at the interface. The term surface-active agent or “surfactant” represents a heterogeneous and long-chain molecule containing both hydrophilic (head) and hydrophobic (tail) moieties (Paria, 2008). They contain a hydrophobic portion with little affinity for the bulk medium and a hydrophilic group attracted to the bulk medium. Consequently, surfactants are used to lower surface tensions and increase solubility. They are used for their detergency power, wetting ability and foaming capacity in petroleum industry (Al-Sabagh et al., 2003), mineral flotation (Asplin et al., 1998; Koopal et al., 1999) and pharmaceutical industries (Madunić-Čačić et al., 2008; Christiansen et al., 2011; Nogueira et al., 2011). But the introduction of surfactants into the environment can lead to contamination concerns. Consequently, the toxicity of the
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surfactant and its potential degradation products needs to be carefully considered prior to the selection of surfactant (Edwards et al., 2003). Biosurfactants are mainly produced by bacteria or yeast, and also available from plants, animals, including human (Paria, 2008). Most microbial surfactants are complex molecules, comprising different structures that include lipopeptides, glycolipids, polysaccharide protein complexes, fatty acids and phospholipids (Thavasi et al., 2008). In the past few decades, biosurfactants have gained more attention and more potentially effective over synthetic surfactants because of their advantages such as high specificity biodegradability and biocompatibility (Torres et al., 2011), low toxicity, ecological acceptability and ability to be produced from renewable and cheaper substrates (Desai and Banat, 1997; Nitschke and Pastore, 2004; Paria, 2008). Therefore, there have more environmental applications in recent years about biosurfactants. Most of the studies focused on their application in the remediation of wastewaters; soils contaminated by heavy metals and hydrophobic organic compounds, such as petroleum hydrocarbons and pesticides (Sponza and Gök, 2010; Zeftawy et al., 2011; Thavasi et al., 2011; Shin et al., 2006; Mulligan and Wang, 2006; Wang and Mulligan, 2009; Cao et al., 2013). A recent study done by Zhou (Zhou et al., 2011) about saponin, a plant-derived non-ionic biosurfactant, described its application in enhancing solubilization of PAHs; it showed that the solubility of PAHs was highly dependent of pH and ionic strength of the solution; the molar solubilization ratio (MSR) of saponin for phenanthrene was about 3–6 times of those synthetic non-ionic surfactants, and decreased about 70% with the increase of solution pH from 4.0 to 8.0, but increased linearly when NaCl concentration increased from 0.01 to 1.0 M; saponin is more effective in enhancing PAH solubilization than synthetic non-ionic surfactants and has potential application in removing organic pollutants from contaminated soils. In this research, we analyzed the structure of a biosurfactant that was produced from oil degradation bacteria Bacillus Lz-2. The corresponding influences of a number of environmental factors (biosurfactant concentrations, pH, ionic strength and temperature) were investigated. The results improve the understanding and prediction of the enhanced solubilization of rhamnolipid biosurfactant for PAHs and provide valuable information for the application of biosurfactants in the remediation of organic contaminated water. The main components of the oil are alkanes, cyclanes and aromatic hydrocarbons. Aromatic hydrocarbons are difficult to be degradated. Rhamnolipid biosurfactant can enhance the solubilization of PAHs in experiment (Zhao et al., 2015), so it can work on other simple components in the oil. Rhamnolipid biosurfactant enhances certain solubilization on oil and achieves certain auxiliary degradation effect. So it can be applied to the marine oil spill.
was 99% similar to Bacillus based on its 16S rRNA gene sequence (1342 bp) and was named Bacillus Lz-2 (GenBank accession number KC256826). The critical micellar concentration (CMC) of this biosurfactant is 240 mg·L−1. The mineral salt medium (MSM) with supplement of sodium citrate 30 g·L−1, peptone 5 g·L−1, NaCl 5 g·L−1; pH = 7, T = 37 °C was used for bacterial growth. For inoculation, the strain was incubated in autoclaved MSM for a week on DHZ-D constant temperature shaker (Taicang Lab-Line Instruments, China) at 150 rpm. After centrifuged at 12,000 r·min− 1 at 4 °C for 20 min, the supernatant was adjusted to pH 2.0 with 1.0 M sulfuric acid, prior to biosurfactant extraction using equal volume of ethyl acetate. The organic phase was separated and the solvent was evaporated to concentrate the biosurfactant. The biosurfactant was then dried using a rotary evaporator. The dried product was washed with absolute ethanol three times completely to remove residual pigments. The IR spectra were recorded on Bruker IFS66 V FT-IR spectrometer (KBr and polyethylene pellets). The FT-Raman and SERS spectra were obtained on a Bruker IFS66V NIR-FT instrument equipped with a FRA 106 Raman module. A Nd/YAG laser at 1064 nm with an output of 300 mW was used for excitation. The detector was a Ge-diode cooled to liquid nitrogen temperature. One thousand scans were accumulated with a total registration time of about 30 min. The spectral resolution was 6 cm−1. HPLC–MS characterization of the biosurfactant was achieved using the quadrupole ion-trap mass spectrometer (1290–6430, Agilent, USA). The scanning type was positive/negative ion scanning with scan range m/z 200–1500. The fragmentor and capillary voltage were set to 110 V and 4.5 kV, and the drying gas temperature was 350 °C with the flow 9 L·min−1. HPLC–MS separation of the rhamnolipid mixtures utilized a reverse phase ZOBARX SB C18 column (5 μm C18, 100 × 2.1 mm, Agilent) and a binary gradient mobile phase comprising HPLC grade H2O as mobile phase A and methanol as mobile phase B. Initially, the proportions were 30% A and 70% B. The flow rate of 0.2 ml min−1 and an injection volume of 1 μL were used throughout.
2. Methods and materials
2.4. PAH detection and quantification
2.1. Chemicals
Detection and quantification of different PAHs (naphthalene, phenanthrene and pyrene) were carried out using Ultraviolet Spectrophotometry (UV2450, Shimadzu).
Naphthalene, phenanthrene and pyrene (purity N98%) were obtained from Aldrich Chemical Company. They were chosen as the three PAH probes because they are petroleum-, coal- and chemical industryrelated organic contaminants that are frequently found in surface and subsurface environments. Dichloromethane and sodium hydroxide were purchased from the Chinese Medicine Group Chemical Reagent Co., LTD. Sulfuric acid was purchased from Tianjin Yao Hua Chemical Reagent Co., LTD. Sodium chloride was used to investigate the effect of ionic strength on PAH solubilization. HCl and NaOH were employed in studying the effect of solution pH on PAH solubilization. 2.2. Purification and identification of the biosurfactant The biosurfactant used in this study was a rhamnolipid surfactant obtained from a bacterial strain, which had been isolated from oil polluted water collected from Dongying, China. The isolated bacteria
2.3. Preparation of standard solution 0.2 g of each PAH compound was dissolved in 100 mL of dichloromethane to prepare a stock solution of 2 g·L−1. The solution was then stored at −10 °C. The PAHs were quantified using 7 levels of external standards obtained by serial dilutions of stock solutions at a concentration range 2–14 mg·L−1 of naphthalene, 0.4–2.8 mg·L− 1 of phenanthrene, 0.2–2.6 mg·L−1 of pyrene.
2.5. Solubilization experiments All experiments were carried out in 100 mL glass conical flasks with Teflon-lined screw caps. The effects of biosurfactants on the solubilization behaviors of naphthalene, phenanthrene and pyrene were examined. For the solubilization experiment, excess PAHs were added to the conical flask and 25 mL background solution. The background solution was comprised of appropriate biosurfactant with 0.01 M NaNO3, which was a biocide system. The pH was adjusted to 7 at a constant ion concentration. The conical flasks were placed on a reciprocal shaker at 30 °C, 100 rpm for 48 h to reach solubilization equilibrium. Preliminary experiments showed that 48 h were enough for the solubilization of phenanthrene to reach equilibrium, and phenanthrene degradation or adsorption by the tubes was negligible. The suspensions
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were then centrifuged at 10,000 rpm for 25 min. Phenanthrenes in the aqueous phase were extracted with dichloromethane, and their concentrations were analyzed by Ultraviolet Spectrophotometry (UV2450, Shimadzu). The UV wavelengths of naphthalene, phenanthrene and pyrene were 228, 254 and 274 nm. All the tests were conducted in triplicate. 2.6. Effect of the biosurfactant concentrations, ionic strength, pH and temperature on the solubilization of naphthalene, phenanthrene and pyrene To investigate the effect of biosurfactant concentrations on the solubilization of naphthalene, phenanthrene, and pyrene, the solubility tests were done using different biosurfactant concentrations (0, 0.112, 0.224, 0.336, 0.448, 0.560 g·L−1). The initial pH value was 7. Then tests were performed in the same manner as the batch solubilization experiments described above. To investigate the effect of ionic strength, the solubility tests were conducted in the presence of biosurfactant and different concentrations of NaCl, which was added at different concentrations (1, 2, 4, 6, 8 and 10 g·L−1) with initial pH = 7. Then the tests were performed in the same manner as the batch solubilization experiments described above. The nature of biosurfactant can vary at different pH. So the solubilization of naphthalene, phenanthrene and pyrene under different pH conditions were studied. Prior to the test, pH of background solution was adjusted to 3, 5, 7, 9, 11 and 13 with the appropriate concentration of HCl and NaOH respectively, the biosurfactant concentration was kept at 0.56 g·L−1. Again, the tests were performed in the same manner as the batch solubilization experiments described above. To investigate the effect of the temperature, reciprocal shakers were respectively set at 25, 30, and 35 °C. The initial concentration for each biosurfactant was 0.56 g·L− 1 with an initial pH of 7. Then the tests were conducted in the same manner as the batch solubilization experiments described above. 3. Results and discussion 3.1. The structure of biosurfactant 3.1.1. FT-IR spectrum There are several literature reports on isolation and biosurfactant production by different species of bacteria. The molecular composition of biosurfactant produced by Lz-2 was evaluated using FT-IR (Fig. 1). The characteristic band at 3386 cm−1 indicates the presence of –OH bonds. Absorption around 2942 cm−1, 2929 cm−1 and 2853 cm−1 is assigned to the symmetric stretch –CH, –CH2 and –CH3 groups of
221
aliphatic chains, and the absorption around 1259 cm−1 and 1371 cm−1 is assigned to the deformation vibrations of –CH. The absorption peak located at 1731 cm−1 indicates the presence of ester carbonyl groups (C=O bond in –COOH). The absorption peak around 1055 cm−1 indicates the presence of polysaccharide or polysaccharide-like substances. The absorption peak at 660 cm−1 indicates the presence of –CH2 group. The above information from the respective wave numbers was confirmed with the glycolipid nature of the biosurfactant. In a previous publication (Aparna et al., 2012), the structure of a rhamnolipid, produced by Pseudomonas sp. 2B was similar to the structure in our research. 3.1.2. HPLC–MS characterization of the rhamnolipid To identify the chemical structure of the product, an aliquot of the purified rhamnolipids produced by Lz-2 was analyzed by HPLC–MS. This analysis revealed 13 homologs with the pseudomolecular ions being between m/z 451 and 809 (Fig. 2). These results were in accordance with the previous studies on rhamnolipids (Manresa et al., 2003; Mulligan et al., 2005; Wray et al., 2007; Paria, 2008; Ibrahim et al., 2010). The presence of rhamnolipid in the crude extract was detected by HPLC–ESI+ MS and HPLC–ESI −MS (Table 1). The main components appeared as sodiated molecular ion clusters in the ESI + MS and [M + Na]+. In the ESI+MS, datum were observed at m/z 453.4 (Rha-C8–C8), 475.3 (Rha-C8–C10, Rha-C10–C8), 679.6 (Rha–Rha-C10– C10 ) and 701.5 (Rha–Rha-C10 –C 12, Rha–Rha-C 12 –C10 ), where Rha—indicates a rhamnose moiety. More detailed information about the structures of the rhamnolipids was derived by ESI −MS, which confirmed the presence of Rha-C8–C8, Rha-C10–C8, Rha-C10– C10, Rha-C 12 –C10, Rha–Rha-C10− C12, Rha–Rha-C12 –C 12. According to the intensities of the fragment ions, the shorter fatty acid is found preferentially directly linked to the rhamnose moiety in LZ-2. The combined data from fragmentation in HPLC–ESI+MS and those from HPLC–ESI −MS allowed the rhamnolipid content of each fraction to be rapidly assessed and showed this combination of techniques to be a very efficient tool for the detailed analysis of complex rhamnolipid mixtures after a simple separation. Therefore, applying LC–MS to rhamnolipid mixtures is an efficient technique because it enables compounds to be identified and chromatographically unresolved pairs of congeners to be quantified. 3.2. The standard curve of the PAHs The standard curves of naphthalene, phenanthrene and pyrene in dichloromethane were measured by UV method. The results were showed in Fig. 3. The standard curves of naphthalene, phenanthrene and pyrene in dichloromethane were Y = 0.108 + 0.05636 ∗ X, R2 = 0.9795; Y = −0.00743 + 0.34723 ∗ X, R2 = 0.9909; Y = −0.02418 + 0.24054 ∗ X, R2 = 0.9941. 3.3. Effect of the biosurfactant concentrations on the solubilization of PAHs
Fig. 1. The FT-IR spectrum of the crude biosurfactant.
Various biosurfactant concentrations were applied to evaluate the performance of biosurfactant in dissolved PAH aqueous phase. As Fig. 4 shows, without the biosurfactant, the concentrations of naphthalene, phenanthrene, and pyrene in aqueous phase were 5.18, 0.9 and 0.28 mg·L− 1. However, in the presence of 0.56 g·L− 1 biosurfactant, they reached 11.91, 2.07 and 1.32 mg·L−1, which corresponds to increase in solubility increased by 129%, 131%, and 372%, respectively. Therefore, the biosurfactant concentration is a critical factor for the solubilization of PAHs. This shows that the presence of biosurfactant has a significant impact on solubilization of PAHs. Upon further experiments, it was observed that PAH solubilization enhanced much more with the increase of biosurfactant concentration (As seen in Fig. 4). When the concentration is above the CMC, hydrophobic pollutants can readily partition into the hydrophobic core at the center of a micelle, thus increasing PAH aqueous concentration through micelle solubilization and promoting solubilization of PAHs.
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Fig. 2. The ESI–MS of the rhamnolipid fractions of purified sample of LZ-2. a) Negative-ion. b) Positive-ion.
The results of the percentage increase still illustrated with the increase of PAH molecular weight, the solubility of the PAHs decreased, but the solubility of percentage increased. Molar solubilization ratio (MSR) (Edwards et al., 1991) in Eq. (1) was described as the ability of PAHs by the biosurfactant. MSR ¼ SPAH; mic −SPAH; cmc =ðC surf −CMCÞ
ð1Þ
Here, Csurf is the concentration of biosurfactant above CMC, SPAH, cmc is the solubility of PAHs at CMC, SPAH, mic is the solubility of PAHs at Csurf. The MSR values were shown in Table 2. Naphthalene has the highest MSR value among the three PAHs, while pyrene has the lowest. In the presence of biosurfactant, PAH solubility decreases as the number of
Table 1 Identification and characterization of rhamnolipid from LZ-2 using HPLC–MS. Major compounds by ESI+MS
Major compounds by ESI−MS
[M + Na] m/z
Substance
[M − H]− m/z
Substance
453.40 475.30 679.60 701.50
Rha-(C8–C8) Rha-(C8–C10) Rha–Rha-(C10–C10) Rha–Rha-(C10–C12)
451.30 487.30 520.30 563.30 583.30 677.50 713.50 740.50
Rha-(C8–C8) Rha-(C8–C10) Rha-(C10–C10) Rha-(C12–C10) Rha-(C12–C12) Rha–Rha-(C10–C10) Rha–Rha-(C10–C12) Rha–Rha-(C12–C12)
+
rings in PAH structure increases. This explains the significance of molecular structure on PAH solubility. 3.4. Effect of ionic strength on the solubilization of PAHs It is well known that ionic strength can affect the micelle aggregation number and CMC values of ionic surfactants. Hence the solubilization capabilities of ionic surfactants for PAHs were examined by changing NaCl concentrations (from 1 to 10 g·L−1) at pH = 7; the results are shown in Fig. 5. As NaCl concentration increases, the solubility of PAHs increases linearly. When the concentration of NaCl jumps from 0 to 2 g·L−1, the solubility of PAHs increases significantly. For the ionic surfactant, adding NaCl electrolyte, CMC of the ionic surfactant is decreased and the logarithm of CMC had a linear relationship with the counter ion concentration values in the system. However, the amplitude of the CMC reducing was decreased as the concentration of NaCl increased beyond the value of 8 g·L− 1. When the concentration of NaCl is 8 g·L−1, the solubility of PAHs reached the maximum (Fig. 5), while the solubility of naphthalene, phenanthrene and pyrene were 2.27, 1.57, and 0.98 mg·L−1 respectively. Due to the presence of electrolyte, the electric double layer of the micelle was compressed, reducing the mutual repulsion between the ionic head surfactant and resulting the surfactant ions into the micelles. The aggregation number greatly increased and the number of the PAHs in the micelle increased too, which was similar with the findings of Zhou et al. (2011). For the non-ionic surfactant, however, the addition of electrolyte at low level does not have obvious effect on the enhanced solubilization of nonionic surfactant for PAHs, but a small change occurs at high electrolyte
S. Li et al. / Marine Pollution Bulletin 101 (2015) 219–225
223
Fig. 4. Different concentrations of surfactant solution on naphthalene, phenanthrene and pyrene solubilization.
(RL) formed either lamella-like structures or lipid aggregates; and for pH N 6.8, the micelles were formed when the rhamnose moiety is negatively charged. Using a transmission electron microscopy, Champion (Champion et al., 1995) observed the RL structure decreasing in size as the pH increased from 5.5 to 8.0. This is due to repulsion between the more negatively charged head groups effectively creating a larger head diameter, and then causing changes in the morphology from lamellar to vesicles to micelles. Based on our results, coupled with the results of Ishigami (Ishigami et al., 1987) and Champion (Champion et al., 1995), we hypothesize that the RL solubility for PAHs increase as the morphology changes when the pH increases from 3 to 11. The results indicated that the solution pH had significant effect on the solubilization capabilities of biosurfactant for PAHs. 3.6. Effect of temperature on the solubilization of PAHs
Fig. 3. The standard curve of a) naphthalene, b) phenanthrene and c) pyrene in dichloromethane.
concentrations due to the “salting out” of the non-ionic surfactants (Paria, 2008). 3.5. Effect of pH on the solubilization of PAHs In general, pH has an obvious effect on the solubilization of ionic surfactants for PAHs. The biosurfactant molecule used in this study is composed of glucuronic acid as part of its head group, and thus the pH has a potential effect on the species of biosurfactant molecule and enhanced solubilization of PAHs. The effect of pH on the solubilization of PAHs in biosurfactant solution was investigated with changing the pH from 3 to 13. With the pH increasing, the solubility of PAHs increased and reached the maximum value of pH 11, as shown in Fig. 6. The corresponding maximum solubility of naphthalene, phenanthrene and pyrene were 4.47, 2.43 and 1.6 mg·L− 1. The micelle structure was changed from flake to vesicular, and finally into micelles, indicating a gradual decrease in its structure. Using fluorescence microscopy, Ishigami (Ishigami et al., 1987) reported that rhamnolipid (RL) form liposome-like vesicles at pH ≤ 6; for pH values of 6–6.6, the rhamnolipid
Temperature has a significant effect on solubilization of organic compounds in the presence of surfactant. For both ionic and nonionic surfactants, an increase in temperature generally results in an increase in the extent of solubilization for both polar solubilization and nonpolar solubilization (Paria, 2008). The effect of temperature on the solubilization of PAHs in the presence of biosurfactant, may be influenced by the characters of the biosurfactant or PAHs. The effect was attributed to (a) the changes in the aqueous phase solubility of the organic compound, and (b) changes in the surfactant micelles with temperature (Yeom et al., 1995). For the ionic biosurfactant, temperature has little effect. However temperature has great influence on the solubility of PAHs. Micelles formed in these aqueous block copolymer systems as the result of warming. Hence, as the temperature of the aqueous system rises, the onset of micelles should provide the mechanism whereby the apparent aqueous solubility of a hydrophobic solute rises dramatically. The impact of temperature on the solubility for naphthalene, phenanthrene and pyrene was shown in Fig. 7. With the temperature increasing, the solubility of phenanthrene and pyrene was increasing, possibly Table 2 The solubility of the PAHs by the biosurfactant. PAHs
Naphthalene Phenanthrene Pyrene
MSR
The solubility of the PAHs/mg·L−1 Pure water
CMC
5.18 0.9 0.28
6.1 1.01 0.65
7.44 2.83 1.34
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S. Li et al. / Marine Pollution Bulletin 101 (2015) 219–225
Fig. 5. Different ionic strengths on naphthalene phenanthrene and pyrene solubilization by surfactant solution.
due to an increase in the number of micelles. When the temperature goes above 30 °C, the solubility of naphthalene decreased which may be caused by the fact that naphthalene volatilized faster with increasing temperature, therefore the solubility of naphthalene in aqueous decreases. With the increase of the ring, the hydrophobic of PAHs increased, the volatile of the PAHs reduced, while the number of micelles increased, thus the solubility of phenanthrene and pyrene increased. The increasing apparent solubility of phenanthrene and pyrene over an extended temperature range suggested a gradual increased in the micellar composition in solution as a result of increasing temperature.
Fig. 7. Different temperatures on naphthalene phenanthrene and pyrene solubilization by surfactant solution.
and 1.6 mg·L−1 when pH was at 11; while the solubility of naphthalene phenanthrene and pyrene were 2.27, 1.57 and 0.98 mg·L−1 when the concentration of NaCl was 8 g·L− 1. The solubility of phenanthrene and pyrene increased with temperature, but the solubility of naphthalene first increased with increasing temperature but then decreased, reaching its maximum solubility at 30 °C. This solubilization switching should be of significant interest to those interested in the technical uses of block co-polymer surfactants. Furthermore, the results from this work also provide a useful assessment tool for rapid selection of biosurfactant conditions for their effectiveness on removing petroleum hydrocarbons from polluted water.
4. Conclusion The results obtained in this study demonstrated the sensitivity of PAH solubilization in micelles of biosurfactant to changes in solution phase pH, temperature and the ionic strength. In particular, the solubility of naphthalene, phenanthrene and pyrene increased linearly with rhamnolipid biosurfactant dose above the biosurfactant CMC. The solubility of PAHs in rhamnolipid biosurfactant solution increased with pH and ionic strength, and reached the maximum value under the pH value of 11 or the NaCl concentration value of 8 g·L− 1. The solubility of naphthalene, phenanthrene and pyrene were 4.47, 2.43
Acknowledgments This research was founded by the “National Natural Science Foundation of China” (41376084); the Major Projects of National High Technology Research and Development Program 863 (2013AA064401); the Public Science and Technology Research Funds Projects of Ocean (201205012); the “Program for Innovative Research Team in University” (IRT1289); and the Open Foundation of Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology of SOA (201402). This is MCTL Contribution No. 104.
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Fig. 6. Different pH on naphthalene, phenanthrene and pyrene solubilization, by surfactant solution.
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