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Nonionic surfactants induced changes in cell characteristics and phenanthrene degradation ability of Sphingomonas sp. GY2B
Ecotoxicology and Environmental Safety 129 (2016) 210–218
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Nonionic surfactants induced changes in cell characteristics and phenanthrene degradation ability of Sphingomonas sp. GY2B Shasha Liu a, Chuling Guo a,b,n, Xujun Liang a, Fengji Wu a, Zhi Dang a,b,n a b
School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, Guangzhou 510006, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 19 December 2015 Received in revised form 26 March 2016 Accepted 28 March 2016 Available online 2 April 2016
Surfactant-mediated bioremediation has been widely applied in decontaminating PAH-polluted sites. However, the impacts of surfactants on the biodegradation of PAHs have been controversial in the past years. To gain a clear insight into the influencing mechanisms, three nonionic surfactants (Tween80, TritonX-100 and Brij30) were selected to systematically investigate their effects on cell surface properties (membrane permeability, functional groups and elements), cell vitality as well as subsequent phenanthrene degradation ability of Sphingomonas sp. GY2B. Results showed that biodegradation of phenanthrene was stimulated by Tween80, slightly inhibited by TritonX-100 and severely inhibited by Brij30, respectively. Positive effect of Tween80 may arise from its role as the additional carbon source for GY2B to increase bacterial growth and activity, as demonstrated by the increasing viable cells in Tween80 amended degradation systems determined by flow cytometry. Although TritonX-100 could inhibit bacterial growth and disrupt cell membrane, its adverse impacts on microbial cells were weaker than Brij30, which may result in its weaker inhibitive extent. Results from this study can provide a rational basis on selecting surfactants for enhancing bioremediation of PAHs. & 2016 Elsevier Inc. All rights reserved.
Keywords: Phenanthrene Surfactant Biodegradation Cell characteristics
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are considered as hazardous pollutants because of their toxicity to human health and ecosystems. Biodegradation has been proved to be one of the most economical and environmentally applicable method for remediation of PAHs-contaminated sites. However, the bioavailability of PAHs has been a major limiting factor for conventional PAHs biodegradation (Vandermeer and Daugulis, 2007). Hence, it is important to seek an alternative clean-up technology that can not only improve PAH bioavailability but also be compatible with biodegradation. Surfactant, due to its ability to enhance the solubility of PAHs and increase their bioavailability (Li and Zhu, 2012; Zhang et al., 2013), has been used for improving PAHs biodegradation and thus developed into surfactant-mediated bioremediation technique (Ahn et al., 2010; Kaczorek et al., 2010). However, in recent years, the negligible even negative effects of surfactants on the biodegradation of PAHs have also been reported (Jin et al., 2007; Mohanty and Mukherji, 2013; Zhang et al., 2013; Zhao et al., 2011). n Corresponding authors at: School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China. E-mail addresses: [email protected] (C. Guo), [email protected] (Z. Dang).
http://dx.doi.org/10.1016/j.ecoenv.2016.03.035 0147-6513/& 2016 Elsevier Inc. All rights reserved.
The degree to which inhibitory and synergistic effects of surfactants depends on the characteristics of surfactants (Li and Zhu, 2012; Zhang et al., 2013; Zhao et al., 2011). Thus it is important to have a better understanding of the interactions between either surfactants and PAHs or surfactants and bacterial cells (Zhang et al., 2013). Most previous studies have focused on the interactions between surfactants and PAHs (e.g. solubilization) (Deshpande et al., 1999; Guha and Jaffé, 1996; Lanzon and Brown, 2013; Zhang and Zhu, 2010). However, the interactions between surfactants and microbial cells alter the characteristics of cell surface, and affect the cell activity as well, which can significantly influence biodegradation of PAHs (Li and Zhu, 2012; Zhang et al., 2013). Therefore, it is crucial to know the biological effects of surfactants on PAHdegrading bacteria to better expound the underlying mechanisms of surfactant-mediated bioremediation. Additionally, in this study, we attempt to introduce flow cytometry, a technique that can monitor cell membrane integrity and discriminate the intact, injured and dead cells accurately at a single-cell level (Soejima et al., 2009), into detecting surfactant induced changes of cell vitality. At present, a variety of surfactants (e.g. ionic, nonionic and biosurfactants) have been used in surfactant-mediated bioremediation trials, among which nonionic surfactants were the most extensively used due to its economic and convenient characters (Jin et al., 2007). However, there are no systematic investigations about
S. Liu et al. / Ecotoxicology and Environmental Safety 129 (2016) 210–218
the effect of nonionic surfactants on bacterial cell properties and subsequent biodegradation of PAHs. Thus, Tween80, TritonX-100 and Brij30, three extensively used nonionic surfactants were selected to investigate (i) their effects on the biodegradation of phenanthrene by Sphingomonas sp. GY2B and (ii) the potential mechanisms based on modifications of microbial surface properties (membrane permeability, functional groups and elements) and subsequent cell vitality. Results from this study can provide effective information to choose surfactants for enhancing bioremediation of PAHs.
2. Materials and methods 2.1. Chemicals Phenanthrene (purity Z98%) purchased from Sigma Aldrich Chemical Company was selected as the representative PAH. Tetraethylene glycol dodecyl ether (Brij30) and t-octylphenoxypolyethoxyethanol (TritonX-100), each with a purity of 99.9%, were obtained from Sigma Aldrich Chemical Company; Ethoxylated sorbitan ester (Tween80, purity Z99.5%) was purchased from Tianjin Kemiou Chemical Reagent Company. Selected physicochemical properties of phenanthrene and surfactants are presented in Table S1 (Supplementary material). The critical micelle concentration (CMC) of Tween 80, TritonX-100 and Brij30 in mineral salt medium (MSM) were 158.4, 125.5 and 14.35 mg/L, respectively. Other reagents were of analytical reagent and purchased from Guangzhou Chemical Reagent Factory, China. Deionized water was used throughout the study. 2.2. Cultivation of Sphingomonas sp. GY2B Sphingomonas sp. GY2B belonging to the Gram-negative bacterium was previously isolated from petroleum-contaminated soil in Guangdong Province, China. The strain was pre-cultivated in the mineral salt medium (MSM) with 100 mg/L phenanthrene as the sole carbon source on a gyratory shaker (150 rpm) at 30 °C for 48 h and used for further studies (Tao et al., 2007). The cultivation solution was spiked by firstly adding an adequate volume of phenanthrene stocked solution (10 g/L) dissolved in acetone to sterilized flasks, and then after evaporation of acetone, autoclaved MSM was added. MSM consisted of the following components (per liter): 5 mL phosphate buffer solution (KH2PO4 8.5 g/L, K2HPO4 H2O 21.75 g/L, Na2HPO4 12H2O 33.4 g/L, NH4Cl 5.0 g/L); 3.0 mL MgSO4 solution (22.5 g/L); 1.0 mL FeCl3 solution (0.25 g/L); 1.0 mL CaCl2 solution (36.4 g/L); 1.0 mL trace element solution (MnSO4 H2O 39.9 g/L, ZnSO4 H2O4 2.8 g/L, (NH4)6Mo7O24 4H2O 34.7 g/L). 2.3. Phenanthrene biodegradation experiments Effects of surfactants on the biodegradation of phenanthrene by GY2B were carried out in 50 mL autoclaved Erlenmeyer flasks. The sterile MSM with surfactants containing 100 mg/L phenanthrene and 2 mL (cell density, 3.6 109 CFU/mL) pre-cultivated GY2B solution were added to the flask and incubated at 30 °C on a gyratory shaker (150 rpm). The concentrations of each surfactant were set as 0.2, 1 and 8 times of their respective CMCs. Control treatments without inoculation were prepared to account for the abiotic loss. Triplicate samples were prepared for each treatment. Samples were taken at selected times to monitor the growth of bacteria and biodegradation of phenanthrene. The bacterial growth was determined by colony-forming units (CFU) plate-counting on nutrient agar as described previously (Wong et al., 2004). The residual phenanthrene in the flasks was extracted with methanol
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and filtered through 0.22 mm Teflon filter units to determine phenanthrene concentrations using an Agilent HPLC (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector and an Agilent Eclipse XDB-C18 column (5 mm, 4.6 mm 150 mm). The HPLC was run with a mobile phase of methanol-water (v/v, 90/10) at a flow rate of 1 mL/min. The UV detector monitored the absorbance at 250 nm (Wei et al., 2015). 2.4. Flow cytometry (FCM) analysis 2.4.1. Sample preparation The samples were drawn from the different reaction systems at 48 h. Then the bacterial cells were collected by centrifugation at 6000 g for 10 min, washed and resuspended in 0.01 M phosphate buffered saline (PBS, pH 7.2; 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4). The cell density was adjusted to optimal values (106 CFU/mL) used for staining test. Non-viable cells were prepared by autoclaving (Zhang and Fang, 2004). 2.4.2. Cell staining The LIVE/DEADs BacLight™ Bacterial Viability kit (Invitrogen, USA) is used in the present study, which is composed of two nucleic acid-binding fluorochrome stains: SYTO9 and Propidium Iodide (PI). SYTO9 is a green fluorochrome that can penetrate all cell (those with intact and damaged membranes) and its affinity for nucleic acids is moderate, while red fluorescent stain PI only penetrates cells with modified membrane permeability or damaged membrane structure and has a higher affinity for nucleic acids (Khan et al., 2010; Soejima et al., 2009). Although SYTO9 can bind to nucleic acids in dead or compromised cells, it will be displaced or its fluorescence reduced by PI; additionally, the membrane of intact cells offers a barrier to entry of PI. Thus, SYTO9/PI could be employed to combine with FCM to monitor cell membrane integrity and then discriminate the intact, injured and dead cells (Biggerstaff et al., 2006; Kramer et al., 2009). Based on this, the bacterial cell viability can be determined. Same volumes of SYTO9 and PI were placed in a microfuge tube and mixed thoroughly by pipetting up and down several times. 3 mL of the dye mixture was added to 1 mL bacterial suspension and incubated at room temperature in the dark for 15 min. The stained cells in the mixture were quantified by FCM. 2.4.3. FCM analysis FCM analysis was performed on a BD FACSAria flow cytometer (Becton Dickinson, USA) equipped with laser excitation. The green fluorescence (from SYTO9) and red fluorescence (from PI) were captured through a 530/30 nm band-pass filter and a 670 nm longpass filter, respectively. Samples were analyzed using a flow rate of 10 mL/min with 10,000 events being acquired for each sample. Then a FACSDiva software was used for data acquisition and analysis (Chen et al., 2014). An unstained bacterial sample (negative control) was employed to confirm that voltages were set appropriately. PI-positive and SYTO9-positive control samples were used to confirm the appropriate areas of stained intact, injured, and dead bacterial populations in dot plot (Fig. S1). Table S2 describes the operating conditions and settings used. 2.5. The morphology of bacterial cells affected by surfactants The morphology of GY2B in the presence and absence of Tween80, TritonX-100 and Brij30 were characterized by scanning electron microscope (SEM) and transmission electron microscope (TEM) as described by Al-Tahhan et al. (2000) and Zhang et al. (2013), respectively.
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spectra were normalized. The FTIR spectra were analyzed using OMNIC software. All spectra were plotted using the same scale on the Y axis (e.g. absorbance).
2.6. Modification of the surface properties of GY2B by non-ionic surfactants The changes of surface properties of cells that exposed to phenanthrene with surfactant concentrations of 0, 0.2, 1 and 8 times of their CMCs, were evaluated by means of cell membrane permeability, cell surface functional groups and element compositions measurements. Each measurement was done in triplicate. 2.6.1. Cell membrane permeability Bacteria can produce β-galactosidase after being activated by lactose, which is a hydrolytic enzyme in the cell membrane. If cell membrane permeability increases, β-galactosidase will release into the culture medium, and hydrolyze o-Nitrophenyl-β-D-Galactopyranoside (ONPG) to galactose and ONP (yellow color), increasing the optical density (OD) of medium (Lehrer et al., 1989). Therefore, effects of surfactants on the membrane permeability of GY2B were evaluated by measuring the release of β-galactosidase activity as described by Zhang et al. (2013). Pre-cultivated bacteria was pipetted into Erlenmeyer flasks containing 100 mL MSM and 2% lactose, and incubated on a rotary shaker (150 rpm) at 30 °C for 12 h. Subsequently, culture were collected by centrifugation (6000 g) for 10 min, washed three times with 0.01 M PBS (pH 7.2) and resuspended (109 CFU/mL) in MSM. Bacterial suspension (2 mL) was mixed with 18 mL surfactant solution at various concentrations (0, 0.2, 1 and 8 CMC) and 1 mL ONPG (30 mM). Sample without surfactants and phenanthrene were used as the blank control. After incubated at 30 °C for 12 h, the culture was centrifuged (12,000 g) for 10 min and the production of o-nitrophenol (ONP) was measured by UV-spectrophotometer at 415 nm (Ibrahim et al., 2000; Zhang et al., 2013). The absorbance of reaction and blank control system were represented by A415,i and A415,0, respectively. The release of β-galactosidase was evaluated based on the productivity of o-nitrophenol (ONP) and calculated using the following formula:
ηONP =
Α 415 × ν ν0 × d × t × ξ
(1)
where A415 (A415 ¼A415,i A415,0) is the absorbance caused by surfactant; ξ is the extinction coefficient (4.86 cm/mM) of ONP; ν, ν0 and t are the sample volume (mL) and reaction time (h), respectively; d is the optical path of cuvette (cm). 2.6.2. Characterization of cell surface element compositions and functional groups The bacteria were harvested from the culture and washed three times with 0.01 M PBS (pH 7.2) to remove all the interfering substances, followed by drying in a freeze drier at 50 °C for 24 h. The dried cells were used to characterize cell surface chemical composition. The element compositions of the dried cells were measured by a X-ray Photoelectron Spectroscopy (XPS, Axis Ultra DLD., Kratos, Britain) having monochromated Al KαX-ray source and a penetration depth of 300 μm (Kim et al., 2015). The binding energy of Cls was shifted to 284.6 eV as an internal reference. Each analysis consisted of a broad survey scan (pass energy 160 eV) for major atomic composition and a high-resolution scan (pass energy 40 eV) for component speciation. The functional groups of the samples were analyzed by Fourier transform infrared (FTIR) spectrometer (Bruker Vertex33,Germany) after the samples were prepared as KBr pellets/discs (Li and Wu, 2010). The samples were recorded between 500 and 4000 wavenumbers under 100 scans with a resolution of 7.5 px 1 (Ojeda et al., 2008). The spectral processing was carried out to remove CO2 and any noise from the spectrum, and finally the
2.7. Statistical analysis The data were analyzed with Origin 8.0. Mean values and standard deviations were calculated. Significances of correlations were determined using Duncan's multiple range test (SPSS 17.0). A value of P o0.05 was considered to be statistically significant.
3. Results and discussion 3.1. Analysis of bacterial cell growth and activity As can be seen from Fig. 1, the growth curve of GY2B reached stable after 24 h of incubation in MSM with phenanthrene. Tween80, TritonX-100 and Brij30 exhibited different impacts on the growth of GY2B during phenanthrene biodegradation. The highest cell density values and maximum specific growth rate were observed in presence of Tween80 (Fig. 1a). Moreover, the higher concentration of Tween80 exerted, the greater positive effect on cell growth occurred, as indicated by the maximum cell density of 9.6 109 CFU/mL at 8 CMC. This was due to the fact that Tween80 can be used as an additional carbon source for GY2B in certain range of concentration (Figs. S4a and S5b–d), which was consistent with the study of Bautista et al. (2009). They found that Tween80 was used as a carbon source by bacteria and thus increased the cell growth rate during PAHs biodegradation. On the other hand, GY2B could not utilize TritonX-100 (Figs. S4b and S5e– g) and Brij30 (Figs. S4c and S5h–j) as the sole carbon source to growth. Moreover, both TritonX-100 (Fig. 1b) and Brij30 (Fig. 1c) exhibited negative influence on cell growth during phenanthrene biodegradation and the inhibitive extent increased with increasing surfactant concentrations. However, the cell growth was more inhibited in the presence of Brij30 than that of TritonX-100, which was further verified at the single cell level determined by FCM measurements. To estimate the proportion of intact, injured and dead cells in samples, FCM measurements were done, as presented in Fig. 2. The results showed that the ratio of intact, injured and dead cells in the presence of surfactants depended on the surfactant concentrations and types. The presence of Tween80 (Fig. 2b–d) increased the percentage of living cells (intact and injured cell), which was consistent with the change of bacterial growth curve (Fig. 1). Compared to the control sample (0 CMC), the proportion of active cells treated with 0.2, 1, and 8 CMC of Tween80 was enhanced by 1.7%, 3.1% and 5.9%, respectively. In addition, Tween80 did not lead to bacterial death (Fig. 2b–d, Fig. S5b–d), which could also be supported by the SEM (Fig. S2b–d) and TEM results (Figs. S3b–d and S6b–d). The bacterial cells were plump and had integral cell structure with uniform cytoplasm and obvious nucleus even when Tween 80 was applied at high concentration (8 CMC). Therefore, the amount of dead cells showed relatively little difference when Tween80 was applied in the range from 0.2 to 8 CMC. Furthermore, Fig. S5b–d exhibited the proportion of intact cells increased with increasing Tween80 concentrations when GY2B grown in MSM only containing Tween80 (without phenanthrene), which also verified that Tween80 can be used as an additional carbon source for GY2B in certain range of concentration, as the previous description in this paper. The proportion of dead cells increased with increasing TritonX100 (0.2–8 CMC) concentrations (Fig. 2e–g and Fig. S5e–g). Similarly, the death rate of cells were also enhanced with Brij30 (0.2–8 CMC) treatments (Fig. 2h–j and Fig. S5h–j). In addition, the ratio of
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Fig. 1. Effects of surfactants on the growth of Sphingomonas sp. GY2B during phenanthrene (100 mg/L) biodegradation. (a) Tween80; (b) TritonX-100; (c) Brij30.
dead cells was higher in the presence of Brij30 than that of TritonX100, indicating that Brij30 was more toxic to bacterial cells than TritonX-100. This was further verified by the SEM (Fig. S2e–j) and TEM (Figs. S3e–j and S6e–j) images. The GY2B cells grown in TritonX100 were shriveled and have a rough surface, along with diffused cytoplasm in core zone of cells, especially at higher concentrations. However, it can be seen that the cell morphology had been affected more severely by Brij30, with more deformed cells and seriously disrupted membrane (Fig. S2h–j). Moreover, the internal structure was also severely damaged under this condition, with more cells ruptured (Figs. S3h–j and S6h–j).The more severely the cells were damaged, the more easily PI entered into cells to combine with DNA/ RNA. This explains why more dead cells were found in Brij30 treated system than TritonX-100 from the FCM results. The aforementioned results agreed well with previous research (Jin et al., 2007) that the toxicity to bacteria cells depended on the polyoxyethylene (POE) chain lengths of non-ionic surfactants, in which the toxicity became lower as the chain length increased. The POE chain lengths of Tween80, TritonX-100 and Brij30 are 20, 9.5 and 4, respectively, which may explain why Brij30 showed the highest toxicity to GY2B in our experiments. Combining the results of the growth curves and single cells characteristics of GY2B, the following phenomena were found: (i) Tween80 has no toxic effect on GY2B in the concentration range of 0.2–8 CMC, and Tween80 can enhance the cell growth and activity of GY2B in the reaction system, even at high concentration (8 CMC); (ii) TritonX-100 and Brij30 can damage the cells and then inhibit cell vitality and bacterial growth; (iii) Brj30 showed more severe negative effect on GY2B than TritonX-100.
3.2. Effect of surfactants on cell membrane permeability of GY2B Effects of Tween80, TritonX-100 and Brij30 on the cell membrane permeability of GY2B were evaluated based on the release of β-galactosidase (Zhang et al., 2013). As shown in Fig. 3, Tween80 did not affect the cell membrane permeability except for high concentration (8 CMC). TritonX-100 exhibited a slightly ability along with Brij30 a notable ability to increase the cell membrane permeability. Tween80 is classified as the heterogeneous surfactant and may not lead to cell membrane lysis (Zhang et al., 2013). Previous researcher (Nazari et al., 2012) has pointed out that heterogeneous surfactant mixes poorly with the fluid membrane lipids and avoid a strong destabilization of lipid bilayer. Failure of the surfactant to mix with the lipid can also drive a segregation of the surfactant from the membrane and thus the cell membrane was little affected (Nazari et al., 2012). Similar result that Tween80 did not obviously affect the cell membrane permeability at the concentration range of 0.2–8 CMC was reported by Zhang et al. (2013). TritonX-100 and Brij30, on the other hand, are the homogeneously surfactants, which can be well miscible with lipids in cell surface (Nazari et al., 2012). As the surfactant concentrations increased beyond the lytic concentration level, surfactant-lipid mixed micelles began to form and gradual solubilization or disintegration of the membrane started (Zhang et al., 2013). As shown in SEM and TEM images (Figs. S2, S3 and S6), GY2B cells presented a much wizened and disorganized membrane as the concentrations of TritonX-100 and Brij30 increased, and even some cell
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Dead cells
Injured cells
Intact cells
Fig. 2. The proportion of intact, injured and dead cells of Sphingomonas sp. GY2B grown on phenanthrene in the absence of surfactants (a) and presence of Tween80 ((b): 0.2 CMC, (c): 1 CMC, (d): 8 CMC), TritonX-100 ((e): 0.2 CMC; (f): 1 CMC; (g): 8 CMC) and Brij30 ((h): 0.2 CMC, (i): 1 CMC, (j): 8 CMC) for 48 h, respectively.
membranes exhibited holes or disruption (where the arrow is pointing) at high concentration (8 CMC). Furthermore, the membrane dissolving and disrupting power of Brij30 (Figs. S2j and S3j) was more severely than that of TritonX-100 (Figs. S2g and S3g), which further verified the higher degree of changes occurred in cell membrane permeability under the treatment of Brij30 than that of TritonX-100.
3.3. Effect of surfactants on cell surface elemental composition and functional groups 3.3.1. XPS spectra XPS can provide information on the atomic elements of the bacterial surface (Kim et al., 2015). Table 1 presents the elemental composition of the control sample and the surfactants-treated
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increase (3.3 fold) of N concentration of Penicillium simplicissimum with the addition of plant-derived biosurfactant saponin has also been found by Liu et al. (2011). However, different result has also been reported (Kim et al., 2015), in which the amount of N was greatly reduced by 20.6%, 31.4%, and 71.1% after the addition of 100, 300 and 500 mg/mL of rhamnolipids. This may be due to the differences in the surfactant types and bacteria species among these studies.
Fig. 3. Effects of Tween80, TritonX-100 and Brij30 on the cell membrane permeability of Sphingomonas sp. GY2B. Significant differences (Po 0.05) from the control (0 CMC) are indicated with “*”.
samples in the form of elemental composition concentration ratios (Na, O, N, Cl and P) with respect to total carbon in GY2B. In general, treatments with Tween80, TritonX-100 and Brij30 decreased the proportions of Na, P and Cl, along with N increased. However, the O/C ratio varied with the types and concentration of surfactants. The addition of Tween80 showed negligible effect on O/C ratios except the 8 CMC treatment system, in which O/C ratios was reduced. The percent of O/C decreased after the addition of 1 and 8 CMC of TritonX-100, but slightly increased at 0.2 CMC. For Brij30, the lower concentrations (0.2 and 1 CMC) decreased and 8 CMC increased the O/C ratios, respectively. This might probably due to the fact that the oxygen originates from two contributions: one attributes to O¼C or P¼O at 530.9 eV belonging to carboxylic acid, carboxylate, ether, carbonyl, amide or phosphoryl groups; the other attributes to hydroxide (C–O–H) or hemiacetal/acetal (C–O– C) at a binding energy around 532.4 eV (Huang et al., 2014; Ojeda et al., 2008). And owing to the complex origins of O detected by XPS, it probably led to the inconsistent changes in different treatments. The nitrogen with a binding energy of 399 eV, which comprises the amine or amide groups (RHN–C¼ O) that are composed of proteins (Huang et al., 2014; Ojeda et al., 2008). As shown in Table 1, the N/C ratio was increased by 3.3–5.2, 1.2–4.7 and 1.8–3.7 fold in the presence of Tween80, TritonX-100 and Brij30, respectively, as compared to the control (without surfactant). Moreover, Tween80-treated samples had the highest N/C ratio, up to 0.034, implying the amount of protein on the cell surface was significantly increased in the presence of Tween80, which was further verified by the FTIR results (Fig. 4a and Fig. S7a). Significant
3.3.2. FTIR spectroscopy FTIR spectra has been widely used to determine cell surface functional groups (Huang et al., 2014; Kim et al., 2015; Ojeda et al., 2008; Zeng et al., 2011). Thus, the changes of functional groups of GY2B with the addition of surfactants were determined by FTIR spectra in the present study. The surface functional groups of GY2B were assigned according to the fowling literatures (Huang et al., 2014; Kim et al., 2015; Ojeda et al., 2008) and summarized in Table S3. Fig. 4 showed the infrared spectrum of GY2B before and after treating with Tween80, TritonX-100 and Brij30 (0.2, 1 and 8 CMC) for 48 h. Results indicated that addition of the three surfactants changed the absorption peak of functional groups. Tween80 (Fig. 4a, Fig. S7a) increased the vibration intensity of some absorbance peaks, especially at 8 CMC. For instance, the adsorption at 2855 cm 1 corresponding to fatty acids and amine peaks (1542 cm 1 and 1652 cm 1) from proteins (Huang et al., 2014; Kim et al., 2015; Ojeda et al., 2008) are stronger than those of the control sample. Distinct absorption regions for polysaccharide (1200–900 cm 1) were also observed. The addition of Tween80 enhanced the intensities of these functional groups, which may contribute to the increase of GY2B growth (Fig. 1) and cell vitality (Fig. 2), and consequently promote phenanthrene biodegradation. Similarly, Zeng et al. (2011) found that the changed FT-IR spectra of cellular envelope might be reason for the enhancement of hexadecane biodegradation in the presence monorhamnolipid. The addition of TritonX-100 (Fig. 4b, Fig. S7b) and Brij30 (Fig. 4c, Fig. S7c) also induced the alterations of some absorbance peaks, including at 2855 cm 1 or 1736 cm 1 corresponding to fatty acid or lipids, 1652 cm 1 (Amide I) and 1542 cm 1 (Amide II) from proteins, 1262 cm 1 (phosphoryl and phosphodiester) associated with nucleic acids, the region between 1200 and 900 cm 1 from polysaccharides, and so on. In particular, 8 CMC of TritonX-100 (Fig. 4b) resulted in the reduction or removal of absorption peak intensities of the fatty acids or lipids from cell membrane (2855 cm 1 or 1736 cm 1). For Brij30 at 8 CMC (Fig. 4c), the peak at 2986 cm 1 and 1736 cm 1 corresponding to fatty acids and lipids disappeared. Reduction in the intensities of the functional groups by TritonX-100 and Brij30 may arise from that they could incorporate into cell membrane, and induce the release of membrane components (e.g. lipids and polysaccharides)
Table 1 The elemental ratios of Sphingomonas sp. GY2B treated with Tween80, TritonX-100 and Brij30. Sample
Phenanthrene (0 CMC) 0.2 CMC Tween80þ phenanthrene 1 CMC Tween80 þphenanthrene 8 CMC Tween80 þphenanthrene 0.2 CMC TritonX-100þ phenanthrene 1 CMC TritonX-100þ phenanthrene 8 CMC TritonX-100þ phenanthrene 0.2 CMC Brij30þ phenanthrene 1 CMC Brij30 þphenanthrene 8 CMC Brij30 þphenanthrene
Elemental composition (molar ratio vs total carbon) Na/C
O/C
N/C
Cl/C
P/C
0.0799 0.0880 0.0416 0.0389 0.0473 0.0485 0.0372 0.0989 0.0314 0.0554
0.1966 0.1990 0.2091 0.1483 0.2507 0.1375 0.1308 0.1621 0.1591 0.2327
0.0055 0.0237 0.0259 0.0341 0.0123 0.0174 0.0312 0.0154 0.0213 0.0256
0.1099 0.1312 0.0525 0.0663 0.0208 0.0512 0.0802 0.1196 0.0225 0.0499
0.0100 0.0097 0 0 0.0023 0 0 0.0048 0.0038 0.0044
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Fig. 4. FTIR spectra of Sphingomonas sp. GY2B grown on phenanthrene in the presence of (a) Tween80, (b) TritonX-100 and (c) Brij30 for 48 h. The concentrations were 0, 0.2, 1 and 8 CMC, respectively.
into the culture medium (Wang et al., 2004). In previous study of our research group (Zhang et al., 2014), disappearances of the absorption peaks corresponding to fatty acids from the cell membrane GY2B might cause the disruption of cell membrane skeleton, leading to an increased membrane permeability or even the disintegration of cell membrane. Boulos et al. (1999) also indicated that cell membrane permeability could be characterized by the leakage of certain molecules (e.g. fatty acids, proteins) from the cell. Thus, we inferred that the modifications of fatty acids or lipids after treating with TritonX-100 and Brij30 (8 CMC) may lead to the increase of membrane permeability or even causing damage to cell membranes. Moreover, owing to the stronger weakening of peak intensity of functional groups by Brij30, it probably indicates that the ability to disrupt cellular membranes of Brij30 is more significantly than TritonX-100, which agreed with the previous results (Section 3.2) in this paper. However, the mechanisms regulating the changes of the functional groups (i.e. fatty acids) still need to be investigated further by other method (e.g. MIDI-MIS). 3.4. Phenanthrene biodegradation by GY2B in the presence of surfactants The effects of Tween80, TritonX-100 and Brij30 on the biodegradation of phenanthrene by GY2B were assessed, as shown in Fig. 5. It can be seen that different modes were obtained under different surfactants. Tween80 (Fig. 5a) enhanced the removal of phenanthrene with increasing surfactant concentrations and reached a maximum at 8 CMC, indicating that the bacteria utilize phenanthrene effectively with the application of Tween80.
Moreover, Tween80 shortened the process of phenanthrene biodegradation, which enhanced the removal efficiencies from 85.7% in the control condition to 95.1%, 99.0% and 99.9% for 0.2, 1 and 8 CMC at 24 h. Similarly, Li and Zhu (2012) found that the phenanthrene removal ratio increased when Tween80 concentrations increased from 0 to 50 mg/L, and reached a maximum of 97.94% at 50 mg/L. It should be pointed out that Tween80 still increased GY2B growth and phenanthrene biodegradation rate when Tween80 added in concentrations over its CMC. Combined with the above analysis of the modifications of cell properties, we deduced that this outcome may stem from the following mechanisms: Tween80 as an additional carbon source of GY2B can enhance the growth of microbial cells with the increasing concentration (Fig. 1) and then increase the viable cell biomass in the reaction systems (Fig. 2b–d, Figs. S5b–d), thereby improving the degradation ability of GY2B (Franzetti et al., 2006). Consistent results was reported by Chen et al. (2013), who found that the potential use of Tween80 as an additional carbon source for the microorganisms may result in higher bacterial growth and pyrene biodegradation rate in the presence of Tween80. Slightly inhibitory effect on phenanthrene removal rate was found with the addition of TritonX-100 (Fig. 5b), while results from Fig. 5c demonstrated that Brij30 dramatically reduced the biodegradation rate in a concentration dependent way. Previous study (In et al., 2001) have suggested positive effects of TritonX100 and Brij30 on phenanthrene biodegradation, likely due to the increase of solubility and bioavailability of phenanthrene. However, in other study (Doong and Lei, 2003) TritonX-100 could decreased pyrene biodegradation rate while Brij30 exhibited
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Fig. 5. Effects of (a) Tween80 (b) TritonX-100 and (c) Brij30 on phenanthrene biodegradation by Sphingomonas sp. GY2B.
negligible effect on pyrene biodegradation. In our study, TritonX100 and Brij30 inhibited phenanthrene biodegradation although the surfactant concentrations were below their respective CMCs. The reason for this phenomenon was presumed to be that TritonX100 and Brij30 were toxic to strain GY2B, specifically: (i) TritonX100 and Brij30 can inhibit bacterial growth, as shown in Fig. 1 and Fig. S4; (ii) the two surfactants can disrupt cell membrane (Figs. S2, S3 and S6) and then enhance the proportion of dead cells (Fig. 2e–j, Fig. S5e–j). The aforementioned phenomenon revealed that TritonX-100 and Brij30 led to the decrease of cell activity or even inactivation of some cells and resulted in biodegradation inhibition. However, the toxicity of Brij30 to GY2B was more significant than that of TritonX-100, causing higher biodegradation rate inhibition than TritonX-100.
biodegradation of PAHs, which will play an important role in choosing suitable surfactants for PAHs biodegradation.
Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2012AA101403), and the Guangdong Provincial Science and Technology Project (2014A020217002).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.03.035.
4. Conclusions This study investigated the effects of three nonionic surfactants (TritonX-100, Tween80 and Brij30) on phenanthrene biodegradation by Sphingomonas sp. GY2B and the modifications of microbial surface properties. Results implied that phenanthrene biodegradation was stimulated by Tween80, which may result from its role as the additional carbon source for GY2B to increase bacterial activity and growth. However, both TritonX-100 and Brj30 were toxic to GY2B as indicated by inhibiting bacterial growth and disrupting cell membrane. Moreover, compared with TritonX-100, Brj30 showed severer toxicity to GY2B, which contributed to its stronger inhibition of phenanthrene degradation by GY2B. Results from this study could provide information to better understand how surfactants affect
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Nonionic surfactants induced changes in cell characteristics and phenanthrene degradation ability of Sphingomonas sp. GY2B Shasha Liu a, Chuling Guo a,b,n, Xujun Liang a, Fengji Wu a, Zhi Dang a,b,n a b
School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, Guangzhou 510006, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 19 December 2015 Received in revised form 26 March 2016 Accepted 28 March 2016 Available online 2 April 2016
Surfactant-mediated bioremediation has been widely applied in decontaminating PAH-polluted sites. However, the impacts of surfactants on the biodegradation of PAHs have been controversial in the past years. To gain a clear insight into the influencing mechanisms, three nonionic surfactants (Tween80, TritonX-100 and Brij30) were selected to systematically investigate their effects on cell surface properties (membrane permeability, functional groups and elements), cell vitality as well as subsequent phenanthrene degradation ability of Sphingomonas sp. GY2B. Results showed that biodegradation of phenanthrene was stimulated by Tween80, slightly inhibited by TritonX-100 and severely inhibited by Brij30, respectively. Positive effect of Tween80 may arise from its role as the additional carbon source for GY2B to increase bacterial growth and activity, as demonstrated by the increasing viable cells in Tween80 amended degradation systems determined by flow cytometry. Although TritonX-100 could inhibit bacterial growth and disrupt cell membrane, its adverse impacts on microbial cells were weaker than Brij30, which may result in its weaker inhibitive extent. Results from this study can provide a rational basis on selecting surfactants for enhancing bioremediation of PAHs. & 2016 Elsevier Inc. All rights reserved.
Keywords: Phenanthrene Surfactant Biodegradation Cell characteristics
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are considered as hazardous pollutants because of their toxicity to human health and ecosystems. Biodegradation has been proved to be one of the most economical and environmentally applicable method for remediation of PAHs-contaminated sites. However, the bioavailability of PAHs has been a major limiting factor for conventional PAHs biodegradation (Vandermeer and Daugulis, 2007). Hence, it is important to seek an alternative clean-up technology that can not only improve PAH bioavailability but also be compatible with biodegradation. Surfactant, due to its ability to enhance the solubility of PAHs and increase their bioavailability (Li and Zhu, 2012; Zhang et al., 2013), has been used for improving PAHs biodegradation and thus developed into surfactant-mediated bioremediation technique (Ahn et al., 2010; Kaczorek et al., 2010). However, in recent years, the negligible even negative effects of surfactants on the biodegradation of PAHs have also been reported (Jin et al., 2007; Mohanty and Mukherji, 2013; Zhang et al., 2013; Zhao et al., 2011). n Corresponding authors at: School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China. E-mail addresses: [email protected] (C. Guo), [email protected] (Z. Dang).
http://dx.doi.org/10.1016/j.ecoenv.2016.03.035 0147-6513/& 2016 Elsevier Inc. All rights reserved.
The degree to which inhibitory and synergistic effects of surfactants depends on the characteristics of surfactants (Li and Zhu, 2012; Zhang et al., 2013; Zhao et al., 2011). Thus it is important to have a better understanding of the interactions between either surfactants and PAHs or surfactants and bacterial cells (Zhang et al., 2013). Most previous studies have focused on the interactions between surfactants and PAHs (e.g. solubilization) (Deshpande et al., 1999; Guha and Jaffé, 1996; Lanzon and Brown, 2013; Zhang and Zhu, 2010). However, the interactions between surfactants and microbial cells alter the characteristics of cell surface, and affect the cell activity as well, which can significantly influence biodegradation of PAHs (Li and Zhu, 2012; Zhang et al., 2013). Therefore, it is crucial to know the biological effects of surfactants on PAHdegrading bacteria to better expound the underlying mechanisms of surfactant-mediated bioremediation. Additionally, in this study, we attempt to introduce flow cytometry, a technique that can monitor cell membrane integrity and discriminate the intact, injured and dead cells accurately at a single-cell level (Soejima et al., 2009), into detecting surfactant induced changes of cell vitality. At present, a variety of surfactants (e.g. ionic, nonionic and biosurfactants) have been used in surfactant-mediated bioremediation trials, among which nonionic surfactants were the most extensively used due to its economic and convenient characters (Jin et al., 2007). However, there are no systematic investigations about
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the effect of nonionic surfactants on bacterial cell properties and subsequent biodegradation of PAHs. Thus, Tween80, TritonX-100 and Brij30, three extensively used nonionic surfactants were selected to investigate (i) their effects on the biodegradation of phenanthrene by Sphingomonas sp. GY2B and (ii) the potential mechanisms based on modifications of microbial surface properties (membrane permeability, functional groups and elements) and subsequent cell vitality. Results from this study can provide effective information to choose surfactants for enhancing bioremediation of PAHs.
2. Materials and methods 2.1. Chemicals Phenanthrene (purity Z98%) purchased from Sigma Aldrich Chemical Company was selected as the representative PAH. Tetraethylene glycol dodecyl ether (Brij30) and t-octylphenoxypolyethoxyethanol (TritonX-100), each with a purity of 99.9%, were obtained from Sigma Aldrich Chemical Company; Ethoxylated sorbitan ester (Tween80, purity Z99.5%) was purchased from Tianjin Kemiou Chemical Reagent Company. Selected physicochemical properties of phenanthrene and surfactants are presented in Table S1 (Supplementary material). The critical micelle concentration (CMC) of Tween 80, TritonX-100 and Brij30 in mineral salt medium (MSM) were 158.4, 125.5 and 14.35 mg/L, respectively. Other reagents were of analytical reagent and purchased from Guangzhou Chemical Reagent Factory, China. Deionized water was used throughout the study. 2.2. Cultivation of Sphingomonas sp. GY2B Sphingomonas sp. GY2B belonging to the Gram-negative bacterium was previously isolated from petroleum-contaminated soil in Guangdong Province, China. The strain was pre-cultivated in the mineral salt medium (MSM) with 100 mg/L phenanthrene as the sole carbon source on a gyratory shaker (150 rpm) at 30 °C for 48 h and used for further studies (Tao et al., 2007). The cultivation solution was spiked by firstly adding an adequate volume of phenanthrene stocked solution (10 g/L) dissolved in acetone to sterilized flasks, and then after evaporation of acetone, autoclaved MSM was added. MSM consisted of the following components (per liter): 5 mL phosphate buffer solution (KH2PO4 8.5 g/L, K2HPO4 H2O 21.75 g/L, Na2HPO4 12H2O 33.4 g/L, NH4Cl 5.0 g/L); 3.0 mL MgSO4 solution (22.5 g/L); 1.0 mL FeCl3 solution (0.25 g/L); 1.0 mL CaCl2 solution (36.4 g/L); 1.0 mL trace element solution (MnSO4 H2O 39.9 g/L, ZnSO4 H2O4 2.8 g/L, (NH4)6Mo7O24 4H2O 34.7 g/L). 2.3. Phenanthrene biodegradation experiments Effects of surfactants on the biodegradation of phenanthrene by GY2B were carried out in 50 mL autoclaved Erlenmeyer flasks. The sterile MSM with surfactants containing 100 mg/L phenanthrene and 2 mL (cell density, 3.6 109 CFU/mL) pre-cultivated GY2B solution were added to the flask and incubated at 30 °C on a gyratory shaker (150 rpm). The concentrations of each surfactant were set as 0.2, 1 and 8 times of their respective CMCs. Control treatments without inoculation were prepared to account for the abiotic loss. Triplicate samples were prepared for each treatment. Samples were taken at selected times to monitor the growth of bacteria and biodegradation of phenanthrene. The bacterial growth was determined by colony-forming units (CFU) plate-counting on nutrient agar as described previously (Wong et al., 2004). The residual phenanthrene in the flasks was extracted with methanol
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and filtered through 0.22 mm Teflon filter units to determine phenanthrene concentrations using an Agilent HPLC (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector and an Agilent Eclipse XDB-C18 column (5 mm, 4.6 mm 150 mm). The HPLC was run with a mobile phase of methanol-water (v/v, 90/10) at a flow rate of 1 mL/min. The UV detector monitored the absorbance at 250 nm (Wei et al., 2015). 2.4. Flow cytometry (FCM) analysis 2.4.1. Sample preparation The samples were drawn from the different reaction systems at 48 h. Then the bacterial cells were collected by centrifugation at 6000 g for 10 min, washed and resuspended in 0.01 M phosphate buffered saline (PBS, pH 7.2; 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4). The cell density was adjusted to optimal values (106 CFU/mL) used for staining test. Non-viable cells were prepared by autoclaving (Zhang and Fang, 2004). 2.4.2. Cell staining The LIVE/DEADs BacLight™ Bacterial Viability kit (Invitrogen, USA) is used in the present study, which is composed of two nucleic acid-binding fluorochrome stains: SYTO9 and Propidium Iodide (PI). SYTO9 is a green fluorochrome that can penetrate all cell (those with intact and damaged membranes) and its affinity for nucleic acids is moderate, while red fluorescent stain PI only penetrates cells with modified membrane permeability or damaged membrane structure and has a higher affinity for nucleic acids (Khan et al., 2010; Soejima et al., 2009). Although SYTO9 can bind to nucleic acids in dead or compromised cells, it will be displaced or its fluorescence reduced by PI; additionally, the membrane of intact cells offers a barrier to entry of PI. Thus, SYTO9/PI could be employed to combine with FCM to monitor cell membrane integrity and then discriminate the intact, injured and dead cells (Biggerstaff et al., 2006; Kramer et al., 2009). Based on this, the bacterial cell viability can be determined. Same volumes of SYTO9 and PI were placed in a microfuge tube and mixed thoroughly by pipetting up and down several times. 3 mL of the dye mixture was added to 1 mL bacterial suspension and incubated at room temperature in the dark for 15 min. The stained cells in the mixture were quantified by FCM. 2.4.3. FCM analysis FCM analysis was performed on a BD FACSAria flow cytometer (Becton Dickinson, USA) equipped with laser excitation. The green fluorescence (from SYTO9) and red fluorescence (from PI) were captured through a 530/30 nm band-pass filter and a 670 nm longpass filter, respectively. Samples were analyzed using a flow rate of 10 mL/min with 10,000 events being acquired for each sample. Then a FACSDiva software was used for data acquisition and analysis (Chen et al., 2014). An unstained bacterial sample (negative control) was employed to confirm that voltages were set appropriately. PI-positive and SYTO9-positive control samples were used to confirm the appropriate areas of stained intact, injured, and dead bacterial populations in dot plot (Fig. S1). Table S2 describes the operating conditions and settings used. 2.5. The morphology of bacterial cells affected by surfactants The morphology of GY2B in the presence and absence of Tween80, TritonX-100 and Brij30 were characterized by scanning electron microscope (SEM) and transmission electron microscope (TEM) as described by Al-Tahhan et al. (2000) and Zhang et al. (2013), respectively.
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spectra were normalized. The FTIR spectra were analyzed using OMNIC software. All spectra were plotted using the same scale on the Y axis (e.g. absorbance).
2.6. Modification of the surface properties of GY2B by non-ionic surfactants The changes of surface properties of cells that exposed to phenanthrene with surfactant concentrations of 0, 0.2, 1 and 8 times of their CMCs, were evaluated by means of cell membrane permeability, cell surface functional groups and element compositions measurements. Each measurement was done in triplicate. 2.6.1. Cell membrane permeability Bacteria can produce β-galactosidase after being activated by lactose, which is a hydrolytic enzyme in the cell membrane. If cell membrane permeability increases, β-galactosidase will release into the culture medium, and hydrolyze o-Nitrophenyl-β-D-Galactopyranoside (ONPG) to galactose and ONP (yellow color), increasing the optical density (OD) of medium (Lehrer et al., 1989). Therefore, effects of surfactants on the membrane permeability of GY2B were evaluated by measuring the release of β-galactosidase activity as described by Zhang et al. (2013). Pre-cultivated bacteria was pipetted into Erlenmeyer flasks containing 100 mL MSM and 2% lactose, and incubated on a rotary shaker (150 rpm) at 30 °C for 12 h. Subsequently, culture were collected by centrifugation (6000 g) for 10 min, washed three times with 0.01 M PBS (pH 7.2) and resuspended (109 CFU/mL) in MSM. Bacterial suspension (2 mL) was mixed with 18 mL surfactant solution at various concentrations (0, 0.2, 1 and 8 CMC) and 1 mL ONPG (30 mM). Sample without surfactants and phenanthrene were used as the blank control. After incubated at 30 °C for 12 h, the culture was centrifuged (12,000 g) for 10 min and the production of o-nitrophenol (ONP) was measured by UV-spectrophotometer at 415 nm (Ibrahim et al., 2000; Zhang et al., 2013). The absorbance of reaction and blank control system were represented by A415,i and A415,0, respectively. The release of β-galactosidase was evaluated based on the productivity of o-nitrophenol (ONP) and calculated using the following formula:
ηONP =
Α 415 × ν ν0 × d × t × ξ
(1)
where A415 (A415 ¼A415,i A415,0) is the absorbance caused by surfactant; ξ is the extinction coefficient (4.86 cm/mM) of ONP; ν, ν0 and t are the sample volume (mL) and reaction time (h), respectively; d is the optical path of cuvette (cm). 2.6.2. Characterization of cell surface element compositions and functional groups The bacteria were harvested from the culture and washed three times with 0.01 M PBS (pH 7.2) to remove all the interfering substances, followed by drying in a freeze drier at 50 °C for 24 h. The dried cells were used to characterize cell surface chemical composition. The element compositions of the dried cells were measured by a X-ray Photoelectron Spectroscopy (XPS, Axis Ultra DLD., Kratos, Britain) having monochromated Al KαX-ray source and a penetration depth of 300 μm (Kim et al., 2015). The binding energy of Cls was shifted to 284.6 eV as an internal reference. Each analysis consisted of a broad survey scan (pass energy 160 eV) for major atomic composition and a high-resolution scan (pass energy 40 eV) for component speciation. The functional groups of the samples were analyzed by Fourier transform infrared (FTIR) spectrometer (Bruker Vertex33,Germany) after the samples were prepared as KBr pellets/discs (Li and Wu, 2010). The samples were recorded between 500 and 4000 wavenumbers under 100 scans with a resolution of 7.5 px 1 (Ojeda et al., 2008). The spectral processing was carried out to remove CO2 and any noise from the spectrum, and finally the
2.7. Statistical analysis The data were analyzed with Origin 8.0. Mean values and standard deviations were calculated. Significances of correlations were determined using Duncan's multiple range test (SPSS 17.0). A value of P o0.05 was considered to be statistically significant.
3. Results and discussion 3.1. Analysis of bacterial cell growth and activity As can be seen from Fig. 1, the growth curve of GY2B reached stable after 24 h of incubation in MSM with phenanthrene. Tween80, TritonX-100 and Brij30 exhibited different impacts on the growth of GY2B during phenanthrene biodegradation. The highest cell density values and maximum specific growth rate were observed in presence of Tween80 (Fig. 1a). Moreover, the higher concentration of Tween80 exerted, the greater positive effect on cell growth occurred, as indicated by the maximum cell density of 9.6 109 CFU/mL at 8 CMC. This was due to the fact that Tween80 can be used as an additional carbon source for GY2B in certain range of concentration (Figs. S4a and S5b–d), which was consistent with the study of Bautista et al. (2009). They found that Tween80 was used as a carbon source by bacteria and thus increased the cell growth rate during PAHs biodegradation. On the other hand, GY2B could not utilize TritonX-100 (Figs. S4b and S5e– g) and Brij30 (Figs. S4c and S5h–j) as the sole carbon source to growth. Moreover, both TritonX-100 (Fig. 1b) and Brij30 (Fig. 1c) exhibited negative influence on cell growth during phenanthrene biodegradation and the inhibitive extent increased with increasing surfactant concentrations. However, the cell growth was more inhibited in the presence of Brij30 than that of TritonX-100, which was further verified at the single cell level determined by FCM measurements. To estimate the proportion of intact, injured and dead cells in samples, FCM measurements were done, as presented in Fig. 2. The results showed that the ratio of intact, injured and dead cells in the presence of surfactants depended on the surfactant concentrations and types. The presence of Tween80 (Fig. 2b–d) increased the percentage of living cells (intact and injured cell), which was consistent with the change of bacterial growth curve (Fig. 1). Compared to the control sample (0 CMC), the proportion of active cells treated with 0.2, 1, and 8 CMC of Tween80 was enhanced by 1.7%, 3.1% and 5.9%, respectively. In addition, Tween80 did not lead to bacterial death (Fig. 2b–d, Fig. S5b–d), which could also be supported by the SEM (Fig. S2b–d) and TEM results (Figs. S3b–d and S6b–d). The bacterial cells were plump and had integral cell structure with uniform cytoplasm and obvious nucleus even when Tween 80 was applied at high concentration (8 CMC). Therefore, the amount of dead cells showed relatively little difference when Tween80 was applied in the range from 0.2 to 8 CMC. Furthermore, Fig. S5b–d exhibited the proportion of intact cells increased with increasing Tween80 concentrations when GY2B grown in MSM only containing Tween80 (without phenanthrene), which also verified that Tween80 can be used as an additional carbon source for GY2B in certain range of concentration, as the previous description in this paper. The proportion of dead cells increased with increasing TritonX100 (0.2–8 CMC) concentrations (Fig. 2e–g and Fig. S5e–g). Similarly, the death rate of cells were also enhanced with Brij30 (0.2–8 CMC) treatments (Fig. 2h–j and Fig. S5h–j). In addition, the ratio of
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Fig. 1. Effects of surfactants on the growth of Sphingomonas sp. GY2B during phenanthrene (100 mg/L) biodegradation. (a) Tween80; (b) TritonX-100; (c) Brij30.
dead cells was higher in the presence of Brij30 than that of TritonX100, indicating that Brij30 was more toxic to bacterial cells than TritonX-100. This was further verified by the SEM (Fig. S2e–j) and TEM (Figs. S3e–j and S6e–j) images. The GY2B cells grown in TritonX100 were shriveled and have a rough surface, along with diffused cytoplasm in core zone of cells, especially at higher concentrations. However, it can be seen that the cell morphology had been affected more severely by Brij30, with more deformed cells and seriously disrupted membrane (Fig. S2h–j). Moreover, the internal structure was also severely damaged under this condition, with more cells ruptured (Figs. S3h–j and S6h–j).The more severely the cells were damaged, the more easily PI entered into cells to combine with DNA/ RNA. This explains why more dead cells were found in Brij30 treated system than TritonX-100 from the FCM results. The aforementioned results agreed well with previous research (Jin et al., 2007) that the toxicity to bacteria cells depended on the polyoxyethylene (POE) chain lengths of non-ionic surfactants, in which the toxicity became lower as the chain length increased. The POE chain lengths of Tween80, TritonX-100 and Brij30 are 20, 9.5 and 4, respectively, which may explain why Brij30 showed the highest toxicity to GY2B in our experiments. Combining the results of the growth curves and single cells characteristics of GY2B, the following phenomena were found: (i) Tween80 has no toxic effect on GY2B in the concentration range of 0.2–8 CMC, and Tween80 can enhance the cell growth and activity of GY2B in the reaction system, even at high concentration (8 CMC); (ii) TritonX-100 and Brij30 can damage the cells and then inhibit cell vitality and bacterial growth; (iii) Brj30 showed more severe negative effect on GY2B than TritonX-100.
3.2. Effect of surfactants on cell membrane permeability of GY2B Effects of Tween80, TritonX-100 and Brij30 on the cell membrane permeability of GY2B were evaluated based on the release of β-galactosidase (Zhang et al., 2013). As shown in Fig. 3, Tween80 did not affect the cell membrane permeability except for high concentration (8 CMC). TritonX-100 exhibited a slightly ability along with Brij30 a notable ability to increase the cell membrane permeability. Tween80 is classified as the heterogeneous surfactant and may not lead to cell membrane lysis (Zhang et al., 2013). Previous researcher (Nazari et al., 2012) has pointed out that heterogeneous surfactant mixes poorly with the fluid membrane lipids and avoid a strong destabilization of lipid bilayer. Failure of the surfactant to mix with the lipid can also drive a segregation of the surfactant from the membrane and thus the cell membrane was little affected (Nazari et al., 2012). Similar result that Tween80 did not obviously affect the cell membrane permeability at the concentration range of 0.2–8 CMC was reported by Zhang et al. (2013). TritonX-100 and Brij30, on the other hand, are the homogeneously surfactants, which can be well miscible with lipids in cell surface (Nazari et al., 2012). As the surfactant concentrations increased beyond the lytic concentration level, surfactant-lipid mixed micelles began to form and gradual solubilization or disintegration of the membrane started (Zhang et al., 2013). As shown in SEM and TEM images (Figs. S2, S3 and S6), GY2B cells presented a much wizened and disorganized membrane as the concentrations of TritonX-100 and Brij30 increased, and even some cell
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Dead cells
Injured cells
Intact cells
Fig. 2. The proportion of intact, injured and dead cells of Sphingomonas sp. GY2B grown on phenanthrene in the absence of surfactants (a) and presence of Tween80 ((b): 0.2 CMC, (c): 1 CMC, (d): 8 CMC), TritonX-100 ((e): 0.2 CMC; (f): 1 CMC; (g): 8 CMC) and Brij30 ((h): 0.2 CMC, (i): 1 CMC, (j): 8 CMC) for 48 h, respectively.
membranes exhibited holes or disruption (where the arrow is pointing) at high concentration (8 CMC). Furthermore, the membrane dissolving and disrupting power of Brij30 (Figs. S2j and S3j) was more severely than that of TritonX-100 (Figs. S2g and S3g), which further verified the higher degree of changes occurred in cell membrane permeability under the treatment of Brij30 than that of TritonX-100.
3.3. Effect of surfactants on cell surface elemental composition and functional groups 3.3.1. XPS spectra XPS can provide information on the atomic elements of the bacterial surface (Kim et al., 2015). Table 1 presents the elemental composition of the control sample and the surfactants-treated
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increase (3.3 fold) of N concentration of Penicillium simplicissimum with the addition of plant-derived biosurfactant saponin has also been found by Liu et al. (2011). However, different result has also been reported (Kim et al., 2015), in which the amount of N was greatly reduced by 20.6%, 31.4%, and 71.1% after the addition of 100, 300 and 500 mg/mL of rhamnolipids. This may be due to the differences in the surfactant types and bacteria species among these studies.
Fig. 3. Effects of Tween80, TritonX-100 and Brij30 on the cell membrane permeability of Sphingomonas sp. GY2B. Significant differences (Po 0.05) from the control (0 CMC) are indicated with “*”.
samples in the form of elemental composition concentration ratios (Na, O, N, Cl and P) with respect to total carbon in GY2B. In general, treatments with Tween80, TritonX-100 and Brij30 decreased the proportions of Na, P and Cl, along with N increased. However, the O/C ratio varied with the types and concentration of surfactants. The addition of Tween80 showed negligible effect on O/C ratios except the 8 CMC treatment system, in which O/C ratios was reduced. The percent of O/C decreased after the addition of 1 and 8 CMC of TritonX-100, but slightly increased at 0.2 CMC. For Brij30, the lower concentrations (0.2 and 1 CMC) decreased and 8 CMC increased the O/C ratios, respectively. This might probably due to the fact that the oxygen originates from two contributions: one attributes to O¼C or P¼O at 530.9 eV belonging to carboxylic acid, carboxylate, ether, carbonyl, amide or phosphoryl groups; the other attributes to hydroxide (C–O–H) or hemiacetal/acetal (C–O– C) at a binding energy around 532.4 eV (Huang et al., 2014; Ojeda et al., 2008). And owing to the complex origins of O detected by XPS, it probably led to the inconsistent changes in different treatments. The nitrogen with a binding energy of 399 eV, which comprises the amine or amide groups (RHN–C¼ O) that are composed of proteins (Huang et al., 2014; Ojeda et al., 2008). As shown in Table 1, the N/C ratio was increased by 3.3–5.2, 1.2–4.7 and 1.8–3.7 fold in the presence of Tween80, TritonX-100 and Brij30, respectively, as compared to the control (without surfactant). Moreover, Tween80-treated samples had the highest N/C ratio, up to 0.034, implying the amount of protein on the cell surface was significantly increased in the presence of Tween80, which was further verified by the FTIR results (Fig. 4a and Fig. S7a). Significant
3.3.2. FTIR spectroscopy FTIR spectra has been widely used to determine cell surface functional groups (Huang et al., 2014; Kim et al., 2015; Ojeda et al., 2008; Zeng et al., 2011). Thus, the changes of functional groups of GY2B with the addition of surfactants were determined by FTIR spectra in the present study. The surface functional groups of GY2B were assigned according to the fowling literatures (Huang et al., 2014; Kim et al., 2015; Ojeda et al., 2008) and summarized in Table S3. Fig. 4 showed the infrared spectrum of GY2B before and after treating with Tween80, TritonX-100 and Brij30 (0.2, 1 and 8 CMC) for 48 h. Results indicated that addition of the three surfactants changed the absorption peak of functional groups. Tween80 (Fig. 4a, Fig. S7a) increased the vibration intensity of some absorbance peaks, especially at 8 CMC. For instance, the adsorption at 2855 cm 1 corresponding to fatty acids and amine peaks (1542 cm 1 and 1652 cm 1) from proteins (Huang et al., 2014; Kim et al., 2015; Ojeda et al., 2008) are stronger than those of the control sample. Distinct absorption regions for polysaccharide (1200–900 cm 1) were also observed. The addition of Tween80 enhanced the intensities of these functional groups, which may contribute to the increase of GY2B growth (Fig. 1) and cell vitality (Fig. 2), and consequently promote phenanthrene biodegradation. Similarly, Zeng et al. (2011) found that the changed FT-IR spectra of cellular envelope might be reason for the enhancement of hexadecane biodegradation in the presence monorhamnolipid. The addition of TritonX-100 (Fig. 4b, Fig. S7b) and Brij30 (Fig. 4c, Fig. S7c) also induced the alterations of some absorbance peaks, including at 2855 cm 1 or 1736 cm 1 corresponding to fatty acid or lipids, 1652 cm 1 (Amide I) and 1542 cm 1 (Amide II) from proteins, 1262 cm 1 (phosphoryl and phosphodiester) associated with nucleic acids, the region between 1200 and 900 cm 1 from polysaccharides, and so on. In particular, 8 CMC of TritonX-100 (Fig. 4b) resulted in the reduction or removal of absorption peak intensities of the fatty acids or lipids from cell membrane (2855 cm 1 or 1736 cm 1). For Brij30 at 8 CMC (Fig. 4c), the peak at 2986 cm 1 and 1736 cm 1 corresponding to fatty acids and lipids disappeared. Reduction in the intensities of the functional groups by TritonX-100 and Brij30 may arise from that they could incorporate into cell membrane, and induce the release of membrane components (e.g. lipids and polysaccharides)
Table 1 The elemental ratios of Sphingomonas sp. GY2B treated with Tween80, TritonX-100 and Brij30. Sample
Phenanthrene (0 CMC) 0.2 CMC Tween80þ phenanthrene 1 CMC Tween80 þphenanthrene 8 CMC Tween80 þphenanthrene 0.2 CMC TritonX-100þ phenanthrene 1 CMC TritonX-100þ phenanthrene 8 CMC TritonX-100þ phenanthrene 0.2 CMC Brij30þ phenanthrene 1 CMC Brij30 þphenanthrene 8 CMC Brij30 þphenanthrene
Elemental composition (molar ratio vs total carbon) Na/C
O/C
N/C
Cl/C
P/C
0.0799 0.0880 0.0416 0.0389 0.0473 0.0485 0.0372 0.0989 0.0314 0.0554
0.1966 0.1990 0.2091 0.1483 0.2507 0.1375 0.1308 0.1621 0.1591 0.2327
0.0055 0.0237 0.0259 0.0341 0.0123 0.0174 0.0312 0.0154 0.0213 0.0256
0.1099 0.1312 0.0525 0.0663 0.0208 0.0512 0.0802 0.1196 0.0225 0.0499
0.0100 0.0097 0 0 0.0023 0 0 0.0048 0.0038 0.0044
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Fig. 4. FTIR spectra of Sphingomonas sp. GY2B grown on phenanthrene in the presence of (a) Tween80, (b) TritonX-100 and (c) Brij30 for 48 h. The concentrations were 0, 0.2, 1 and 8 CMC, respectively.
into the culture medium (Wang et al., 2004). In previous study of our research group (Zhang et al., 2014), disappearances of the absorption peaks corresponding to fatty acids from the cell membrane GY2B might cause the disruption of cell membrane skeleton, leading to an increased membrane permeability or even the disintegration of cell membrane. Boulos et al. (1999) also indicated that cell membrane permeability could be characterized by the leakage of certain molecules (e.g. fatty acids, proteins) from the cell. Thus, we inferred that the modifications of fatty acids or lipids after treating with TritonX-100 and Brij30 (8 CMC) may lead to the increase of membrane permeability or even causing damage to cell membranes. Moreover, owing to the stronger weakening of peak intensity of functional groups by Brij30, it probably indicates that the ability to disrupt cellular membranes of Brij30 is more significantly than TritonX-100, which agreed with the previous results (Section 3.2) in this paper. However, the mechanisms regulating the changes of the functional groups (i.e. fatty acids) still need to be investigated further by other method (e.g. MIDI-MIS). 3.4. Phenanthrene biodegradation by GY2B in the presence of surfactants The effects of Tween80, TritonX-100 and Brij30 on the biodegradation of phenanthrene by GY2B were assessed, as shown in Fig. 5. It can be seen that different modes were obtained under different surfactants. Tween80 (Fig. 5a) enhanced the removal of phenanthrene with increasing surfactant concentrations and reached a maximum at 8 CMC, indicating that the bacteria utilize phenanthrene effectively with the application of Tween80.
Moreover, Tween80 shortened the process of phenanthrene biodegradation, which enhanced the removal efficiencies from 85.7% in the control condition to 95.1%, 99.0% and 99.9% for 0.2, 1 and 8 CMC at 24 h. Similarly, Li and Zhu (2012) found that the phenanthrene removal ratio increased when Tween80 concentrations increased from 0 to 50 mg/L, and reached a maximum of 97.94% at 50 mg/L. It should be pointed out that Tween80 still increased GY2B growth and phenanthrene biodegradation rate when Tween80 added in concentrations over its CMC. Combined with the above analysis of the modifications of cell properties, we deduced that this outcome may stem from the following mechanisms: Tween80 as an additional carbon source of GY2B can enhance the growth of microbial cells with the increasing concentration (Fig. 1) and then increase the viable cell biomass in the reaction systems (Fig. 2b–d, Figs. S5b–d), thereby improving the degradation ability of GY2B (Franzetti et al., 2006). Consistent results was reported by Chen et al. (2013), who found that the potential use of Tween80 as an additional carbon source for the microorganisms may result in higher bacterial growth and pyrene biodegradation rate in the presence of Tween80. Slightly inhibitory effect on phenanthrene removal rate was found with the addition of TritonX-100 (Fig. 5b), while results from Fig. 5c demonstrated that Brij30 dramatically reduced the biodegradation rate in a concentration dependent way. Previous study (In et al., 2001) have suggested positive effects of TritonX100 and Brij30 on phenanthrene biodegradation, likely due to the increase of solubility and bioavailability of phenanthrene. However, in other study (Doong and Lei, 2003) TritonX-100 could decreased pyrene biodegradation rate while Brij30 exhibited
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Fig. 5. Effects of (a) Tween80 (b) TritonX-100 and (c) Brij30 on phenanthrene biodegradation by Sphingomonas sp. GY2B.
negligible effect on pyrene biodegradation. In our study, TritonX100 and Brij30 inhibited phenanthrene biodegradation although the surfactant concentrations were below their respective CMCs. The reason for this phenomenon was presumed to be that TritonX100 and Brij30 were toxic to strain GY2B, specifically: (i) TritonX100 and Brij30 can inhibit bacterial growth, as shown in Fig. 1 and Fig. S4; (ii) the two surfactants can disrupt cell membrane (Figs. S2, S3 and S6) and then enhance the proportion of dead cells (Fig. 2e–j, Fig. S5e–j). The aforementioned phenomenon revealed that TritonX-100 and Brij30 led to the decrease of cell activity or even inactivation of some cells and resulted in biodegradation inhibition. However, the toxicity of Brij30 to GY2B was more significant than that of TritonX-100, causing higher biodegradation rate inhibition than TritonX-100.
biodegradation of PAHs, which will play an important role in choosing suitable surfactants for PAHs biodegradation.
Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2012AA101403), and the Guangdong Provincial Science and Technology Project (2014A020217002).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.03.035.
4. Conclusions This study investigated the effects of three nonionic surfactants (TritonX-100, Tween80 and Brij30) on phenanthrene biodegradation by Sphingomonas sp. GY2B and the modifications of microbial surface properties. Results implied that phenanthrene biodegradation was stimulated by Tween80, which may result from its role as the additional carbon source for GY2B to increase bacterial activity and growth. However, both TritonX-100 and Brj30 were toxic to GY2B as indicated by inhibiting bacterial growth and disrupting cell membrane. Moreover, compared with TritonX-100, Brj30 showed severer toxicity to GY2B, which contributed to its stronger inhibition of phenanthrene degradation by GY2B. Results from this study could provide information to better understand how surfactants affect
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