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Mechanism of enhancing pyrene-degradation ability of bacteria by layer-by-layer assembly bio-microcapsules materials
Ecotoxicology and Environmental Safety 181 (2019) 525–533
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Mechanism of enhancing pyrene-degradation ability of bacteria by layer-bylayer assembly bio-microcapsules materials
T
Fucai Denga,b, Jianteng Suna, Rongni Doua,b, Wangming Denga, Yi Liua, Chen Yangb,∗, Zhi Dangb,∗ a
Guangdong Provincial Key Laboratory of Petrochemical Pollution Processes and Control, School of Environmental Science and Engineering, Guangdong University of Petrochemical Technology, Maoming, Guangdong, 525000, China b College of Environment and Energy, South China University of Technology, Guangzhou, 510006, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Layer-by-layer assembly Bio-microcapsules Flow cytometry analysis Extracellular polymeric substances Laser light scattering Fluorescence quenching
The mechanism of improving pyrene (PYR)-degrading ability of bacteria CP13 in Layer-by-layer (LBL) assembly chitosan/alginate (CHI/ALG) bio-microcapsules was investigated. Flow cytometry analysis showed that LBL microcapsules could effectively slow down the increasing rate of bacterial cell membrane permeability and the decreasing rate of the membrane potential, so as to reduce the death rate and number of the cells, which could protect the degrading bacteria. The results of Fluorescence spectrum, circular dichroism (CD) spectrum and laser light scattering (LLS) analysis revealed that the other possible mechanism for LBL microcapsules to promote bacterial degradation were following: CHI could enter the secondary structure of the protein of the extracellular polymeric substances (EPS) from CP13 and combined with EPS to generate a stable ground material, which had larger molecular weight (3.76×106 g mol-1) than the original EPS (2.52×106 g mol-1). The combination of CHI and EPS resulted in the decrease of the density of EPS from 1.18 to 0.72 g L-1, suggesting that CHI can loosen the EPS configurations, improving the capture ability of bacteria for PYR as well as the mass transfer of PYR from the extracellular to intracellular, thus eventually promoting the bacteria degrade performance.
1. Introduction Bacterial immobilization methods have been commonly employed to enhance the bioremediation of organic pollutants, including the physical adsorption of bacteria on carriers (Biswas et al., 2016; Lin et al., 2010; Liu et al., 2012) and entrapment of bacteria in alginate beads (El-Naas et al., 2009; Tan et al., 2014; Zhang et al., 2008). Compared to the free cells, immobilization has inherent advantages including enhanced stability of the system, easy separation of cells, minimizing or eliminating the cell contaminations in the products, convenient recovery and re-use of cells which enable their frequent use in the process (Zhang et al., 2010). In our previous studies, Layer-by-layer (LBL) assembly chitosan/ alginate (CHI/ALG) bio-microcapsules immobilized bacteria CP13 could gain a much higher tolerance to environmental stress (i.e. high initial pyrene (PYR) concentration, extremely low pH values or temperatures and high salt stress) (Deng et al., 2017). LBL bio-microcapsules also could cause a significant increase in the biodiversity of the bacterial community in the PYR-contaminated soil, successfully accounting for accelerated PYR removal (Deng et al., 2016). However, the mechanism was not clear yet. The strategy of bacteria immobilization
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to enhance PYR degradation could be attributed to the protective effect provided by the bio-microcapsule for the bacteria from the nocuity causing by the pollutant. On the other hand, the interaction which may occur between bio-microcapsules materials (CHI or ALG) and the extracellular polymeric substances (EPS) of the bacteria may be another explanation for the accelerated rate of PYR-degrading. EPS is a complex high-molecular-weight mixture of polymers produced by microorganisms, (Sheng et al., 2010). Many organic compounds, such as phenanthrene, benzene and dyes (e.g., toluidine blue), can be absorbed by EPS due to the existence of some hydrophobic regions in EPS (Liu et al., 2001; Sheng et al., 2008; Späth et al., 1998). The adsorption capacity of microbial cells decreased significantly after that EPS were removed (Khunjar and Love, 2011). Some organic polymer composite might be formed between EPS and compounds through hydrophobic interaction, hydrogen bond or electrostatic interaction (Clara et al., 2004; Lindberg et al., 2005; Yang et al., 2011), which may affect the physical and chemical properties of the EPS, resulting in the change of the degrading ability of microbial cells. However, the effect mechanism of the interaction between organic polymer composite and the EPS on the degrading ability of the cells is not clear, due to the lack of appropriate analytical tools to identify the complex interactions between them.
Corresponding authors. College of Environment and Energy, South China University of Technology, Guangzhou, China. E-mail addresses: [email protected] (F. Deng), [email protected] (C. Yang), [email protected] (Z. Dang).
https://doi.org/10.1016/j.ecoenv.2019.06.016 Received 25 February 2019; Received in revised form 24 May 2019; Accepted 5 June 2019 Available online 21 June 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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Cells, thermo fisher scientific (USA)) was used to stain the collected cells to analyze the ratio of dead, damaged and live cells of the free and LBL microencapsulated bacteria through flow cytometry, respectively. The experimental processes are as follows: firstly, the CP13 cells were collected approximately 5×106 cells mL-1. After washed 2 to 3 times with PBS, the cells was mixed with SYTO 9/PI dye solution (3 μL in proportion of 1:1) and incubated for 15 min avoiding light at room temperature. Then, the cell solution was passed through 400 mesh nylon screen, and carried out flow cytometry detection (Soejima et al., 2009). The detection excitation light was 488 nm, SYTO 9 dye was recorded on FL1 detection channel, PI dye was detected on FL3 channel, and more than 10,000 cells were counted.
In this study, the protection effects provided by the bio-microcapsules for the bacteria as well as the interactions between CHI/ALG and EPS were investigated. A combined use of fluorescence spectrum analysis, circular dichroism (CD) spectrum analysis and laser light scattering (LLS) was employed to elucidate such interaction. This study aimed to: (1) explore the protection for the cells by the LBL bio-microcapsules; (2) investigate the mechanisms for the interaction between CHI/ALG and EPS and its effect mechanism on the enhancing degrading ability of the cells. The results might favor a better understanding about the effect mechanism of cell immobilization technology on improving the cell’s degrading ability. 2. Materials and methods
2.3. EPS interacts with LBL microcapsule materials 2.1. Materials and organism 2.3.1. Extraction of EPS and its combination with CHI or ALG EPS from CP13 cells was extracted according to an improved thermal extraction method (Li and Yang, 2007). Firstly, the centrifuged cells were resuspended in 0.05% sodium chloride solution and then keep in water bath at 60 °C for 30 min. Then the bacteria solution was centrifuged for 15 min at 10000 rpm. EPS in the upper solution was collected and passed through the 0.45 μm cellulose acetate membrane. Then, EPS was further purified with 5000 Da dialysis bag to remove ions and small molecules. Finally, EPS was freeze-dried and stored in the dryer for standby. The samples for analysis the interaction between EPS and microencapsulated materials (CHI and ALG) were prepared in 50 mM (pH 7.0) PBS using 20 mL glass tube. Different volume of CHI or ALG solution was added to the tube containing 5 mL 200 mg L-1 EPS. Then, PBS was added to the mixture to make the total volume be 10 mL. Before analysis, the solution were mixed well using a vortex oscillator and balanced for 4 h.
The microorganism used in this study was Mycobacterium gilvum CP13, which was isolated from the activated sludge of a coking plant in Shaoguan, Guangdong, China (Wu et al., 2014). The bacteria were cultured in fresh nutrient broth (aqueous solution; 1.8%) and collected according to the method of Deng et al. (2017). 2.2. The protective effect of LBL microcapsules on bacteria 2.2.1. Determination of the permeability of the bacteria membrane In this study, the CP13 cells were dyed by propidium iodide (PI), and the membrane permeability of the cells are tested to figure out the characteristic changes of the CP13 cell surface. During the experiment, the CP13 cells were collected approximately 5×106 cells mL-1 during the PYR degradation process (0, 3, 5, 7d), PYR degradation using LBL bio-microcapsules was conducted in batch experiments in 100-ml shake flasks containing 20 ml of mineral salt medium (MSM) with PYR as the sole carbon source at 30°C and 180 rpm avoiding light. The MSM consisted of 2.5 g L-1 K2HPO3, 0.77 g L-1 KH2PO4, 100 mg L-1 (NH4)2SO4, 20 mg L-1 MgSO4·7H2O, 10 mg L-1 CaCl2·2H2O, 1.2 mg L-1 FeSO4·7H2O, 0.3 mg L-1 MnSO4·H2O, 0.3 mg L-1 ZnSO4·7H2O, and 0.1 mg L-1 CoSO4·7H2O, 0.1 mg L-1 (NH4)6Mo7 O24·4H2O. The initial PYR concentration was 50 mg L-1. After washing the cells for 2 to 3 times with phosphate buffer solution (PBS), they were fixed with 1% paraformaldehyde and saved under 4 °C for further analysis (Verthé and Verstraete, 2006). Before performing flow cytometry, the cells must be washed with PBS 2 to 3 times to remove the redundant paraformaldehyde. After that, enough PI solution was added to make the PI concentration as 50 μg mL-1 and the bacteria was hatched for 30 min at 37°C in the absence of lights. Finally, the bacteria solution was passed through 400 mesh nylon mesh screen, then poured into the Beckman Coulter XCL-MCL flow cytometry analysis (the test excitation light is 488 nm, FL3 detector test, 605-635 nm emission light intensity) (Günther et al., 2007).
2.3.2. Determination of bacterial EPS components (1). Polysaccharide content was analyzed using phenol-sulphate acid method (Dubois et al., 1956): 0.1 g of glucose was dissolved in 100 mL ultrapure water to obtain a standard glucose solution. 0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.4 mL standard glucose solution was added to 10 mL colorimetric tube respectively. Ultrapure water was supplemented to the volume of 1.0 mL and then 1 mL of phenol solution (5%) added and shaken well. Then 5 mL of concentrated sulfuric acid was put in and placed in a constant temperature water bath of 96°C for 20 min. When the mixture was cooled down to room temperature, the light absorbance was tested on UV spectrophotometer at the wavelength of 490 nm. The standard curve of polysaccharides was obtained with the concentration of polysaccharide as the horizontal and the absorbance as vertical coordinates, respectively. The light absorbance of 0.1 mL EPS solution (l g L-1) was determined using the same operation process. The content of polysaccharide in EPS was calculated through the standard curve. (2). The content of protein in EPS was measured by coomassie brilliant blue method with spectrophotometer under the wavelengths of 595 nm. (3). Humic acid was determined using the standard curve method and determining by UV spectrophotometer.
2.2.2. Determination of membrane potential In this study, Rhodamine 123 (Rh123) was used as the stain agent, and the cell membrane potential of free and microcapsule immobilized bacteria was quantitatively analyzed by flow cytometry during the process of PYR degradation. Rh-123 was made up to 1 mg mL-1 in ethanol and maintained in −20oC as the stain stock solution. The working concentration of Rh-123 was 10 mg mL-1 100 mL of the cell suspension was added to 400 mL of the stain in a 5 mL polypropylene tube. The mixture was incubated in a dark room at the ambient temperature for 10 min. Then, measurements were carried out using a Beckman Coulter XCL-MCL flow cytometry analysis with fluorescence detector, which detected appropriately filtered light at green (FL1, 525 nm). A total number of 10,000 cells were recorded for each sample.
The chemical structures of the EPS components were also characterized by a Fourier transform infrared (FT-IR) spectrophotometer. 2.3.3. LLS analysis LLS analysis was performed on an ALV/DLS/SLS-5022F spectrometer. Three samples (500 mg L-1 EPS, 500 mg L-1 EPS +250 mg L-1 CHI/ALG, 500 mg L-1 EPS + 500mg L-1 CHI/ALG) which prepared by 50 mm PBS (pH= 7.0) were used, and the temperature of the test was
2.2.3. Determination of the survival and damage of the bacteria SYTO 9/PI dye (LIVE-or-DIE™ Viability/Cytotoxicity Kit for Animal 526
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3.1.2. The change of cell membrane potential The changes in cell membrane potential of CP13 bacteria in free form and LBL microcapsules were shown in Fig. 2 and Table S2. It can be seen from the results that the fluorescence signal of Rh123 in the samples increased gradually, indicating that the cell membrane potential was decreased. The decrease rate of fluorescence signal in the free bacteria was faster than that of LBL microcapsules. Specifically, the fluorescence intensity of free cells decreased from the initial 141.25 to 63.10 in the 3rd day, and eventually to 3.16 in the 7th day, while the intensity value of immobilized one was still above 19 after degradation to the 7th day. Under normal circumstances, the potential of the cell membrane is negative inside and positive outside. After being stimulated, some cell membranes become positive inside and negative outside, which reduces the negative value of the membrane potential. Studies have showed that changes in cell membrane potential were closely related to changes in Na+ and K+ concentrations in- and outside the cells (Volkov et al., 2011). When cell apoptosis occurs, the integrity of the cell membrane is destroyed, the cell membrane permeability increases (Chen et al., 2014), the intracellular Na+ is gradually released into the cell, and the extracellular K+ is continuously transported to the cell via the K+-ATPase ion pump, which results in intracellular high K+ and low Na+ and extracellular environment high Na+ and low K+, eventually leading to decreased cell membrane potential. Therefore, the change of membrane permeability is the key point of cell apoptosis. In this study, the signal of Rh123 dye in the LBL microcapsule immobilized bacteria system was 6.31 times higher than the free one after 7 days of degradation, which showed that the immobilization treatment of the microcapsules slowed down the damage rate of the cell membrane integrity and the increase rate of membrane permeability. These results further confirmed that microcapsule had a protective effect on the cells, which may be the possible mechanism of the enhancement PYR degradation.
25.0 ± 0.1°C. The physical parameters of EPS were measured by dynamic light scattering and static light scattering, including the average hydraulic radius (< Rh >), z-direction mean square rotation radius (< Rg >) and apparent average molecular weight (Mw). Among them, the internal density (C*) can be calculated from Mw and < Rg > (Wang et al., 2012a). 2.3.4. Spectrum analysis The wavelength of synchronous fluorescence spectrum analysis was from 240 to 300 nm, and the excitation emission interval was 60 nm. Circular dichroism spectrum analysis was performed on a circular dichroism spectroscopy. The EPS spectra with different concentrations of CHI or ALG were recorded from 190 to 240 nm and scanned three times. Three-dimensional excitation–emission matrix (3D-EEM) fluorescence was carried out by three-dimensional fluorescence spectrophotometer (F7000, Hitachi, Japan). Excitation wavelength was 200 to 400 nm with an interval of 10 nm. Emission wavelength was 300 to 550 nm with scanning interval 0.5 nm. Ultra-pure water was scanned as a background. Specific operations refer to the literature (Li et al., 2008). EEM date is analyzed by MATLAB software. The UV absorption spectrum was analyzed by UV spectrophotometer with 1 cm cell and the wavelength range was 200 to 800 nm. 3. Results and discussion 3.1. Protective effect of LBL microcapsule on bacteria 3.1.1. The change of membrane permeability of bacteria The first step of the degradation process is the contact between microorganism and pollutants. The degradation ability of bacteria is affected at the contact step, due to the toxicity of pollutants on the bacterial cell wall and plasma membrane, which could change the microbial membrane permeability and cell membrane electric potential. It is pointed out that when the lgKow of organic solvent in contact with bacteria is 1.5–3, the fatty acid composition of bacterial cell membrane will be changed, destroying the structure and permeability of cell membrane and causing a lot of dissolution and death of bacteria, and eventually leading to the decrease of degradation rate (Shi et al., 2007). In order to explore the protective effect of LBL microcapsules for the bacteria CP13 in PYR degradation process, the changes of membrane permeability during the PYR degradation with initial concentration of 50 mg L-1 were studied in the presence and absence of microcapsules conditions. The results (Fig. 1 and Table S1) showed that during the progress of PYR degradation in both treatments, the signal of PI dye was gradually increased, indicating that the membrane permeability of the bacteria was gradually enhanced. At the initial stage, the main cells in the system were live with intact membrane structure and low permeability. The PI dye of the large molecule cannot enter the cell, and the PI signal was very weak at this stage. With the degradation time extension, the signal strength of PI detection increased, suggesting more PI molecules entered the cell and bound with DNA of bacteria. It could be concluded the integrity of the bacterial cell membrane damaged gradually, leading to the stronger permeability of cell membrane. The signal intensity of PI obtained from free bacterial treatment increased significantly faster than that of LBL immobilized bacteria, from the initial 1.41 to 22.39 on the 3rd day, and then to 50.12 on the 5th day, and finally increased to 63.10 on the 7th day. This indicates that, under the high initial concentration of PYR, the effect of PYR on the cell membrane of free bacteria leads to rapid destruction of membrane structure and strong penetration of cell membrane. After 7 days of degradation, the fluorescence intensity of the PI dye in the system was still lower than 18, which was about 1/4 of the free bacterial system, demonstrating that LBL microcapsules slowed down the damage rate of cell membrane integrity, manifesting a certain protective effect on cells.
3.1.3. The change in cell survival and damage The activity of free and LBL microcapsules immobilized CP13 cells was detected during 50 mg L-1 PYR degradation process (Fig. 3). It’s found that four groups of cells (A1, A2, A3, and A4) appeared, which were dead cells labeled by PI (red), damaged cells labeled by PI and SYTO 9 (blue), live cells labeled by SYTO 9 (green), and negative cells unlabeled (black), respectively. In the scatter plot, the changes of the proportion of the four groups of cells can directly reflect the inhibitory action of PYR on CP13 bacteria. Fig. 3 showed that excessive PYR can induce cell apoptosis during the degradation process. This may be related to the changes of functional group on cell surface, enhancing surface hydrophobicity (Li and Zhu, 2012), thereby promoting the contact of PYR with the cell surface and the migration of PYR into intracellular. When the excessive PYR enters the cell, it would produce further damage to the cell. The survival and damage of the cells treated with free and LBL microcapsule immobilized were listed in Tables S3 and S4. As can be seen from the tables, the proportion of active cells gradually decreased with the extension of the degradation time. The proportion of live cells in free bacteria decreased faster than LBL immobilized bacteria, from the 100% (initial) to 83.64% (3rd d), and then to 68.43% (5th d), and finally to 46.14% (7th d). However, the proportion of LBL immobilized bacteria in the active cells can still be remained at more than 72% at the 7th day. The results further indicated that at the high initial PYR concentration (50 mg L-1), the effect of PYR on the cell membrane of free bacteria is obvious, leading to membrane structure damage, which makes the cell death and the degradation of PYR more slowly. However, LBL immobilization treatment can make this damage effect weakened, and the rate of cell death decreased, thus make the cells showed a higher PYR degradation rate.
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Fig. 1. Cell permeability quantified using flow cytometry in free bacteria (FB) and LBL systems (The initial PYR concentration was 50 mg L-1)
degradation bacteria CP13 were shown in Fig. S1 and Table S6. The infrared spectrum exhibited a strong absorbance at 2929 cm−1, which represents C–H stretching vibrations (Caroni et al., 2012; Wang et al., 2012b). It was found that the characteristic peaks of protein in EPS appeared at 1635 and 1298 cm-1, corresponding to NH (1) (Wei et al., 2016) and amino (3) in the protein in EPS, respectively (Chen et al., 2013). The band near 1400 cm−1 was related to the absorption of C]O symmetric stretching vibration in the carboxylic groups (Chen et al., 2013; Wei et al., 2016). In addition, peaks at 1240 cm-1 can also be attributed to the C–N stretching vibration in the protein (Chen et al., 2013). Various characteristic functional groups of polysaccharide were also shown in the spectrum chart. For example, 1072 cm-1 and 1056 cm1 peaks attributed to the C–O, C–C stretching vibration (Song et al., 2014) and C–O–C, C–O–H deformation vibration of the polysacchaide, respectively, which confirms the presence of a certain amount of polysaccharide in EPS (Chen et al., 2013). In conclusion, the results of infrared characterization showed that the components of EPS produced by the CP13 bacteria have typical protein, polysaccharide characteristic spectrum (Chen et al., 2013), which verified the results of the components analysis in proportion 3.2.1.
3.2. Interaction between bacterial EPS and LBL microcapsules 3.2.1. Components analysis of the EPS The composition analysis of EPS produced by CP13 bacteria were shown in Table S5. Results showed the main component of the EPS was protein, accounting for about 70%, and followed by humic acid and polysaccharide, which were consistent with the previous studies (Gu et al., 2017; Pellicer-Nàcher et al., 2013). Similarly, when Zhang et al. (2014) extracted EPS from a PAH-degrading bacterium by four methods including cation exchange resin method, ultrasonic method, ultrasoniccation exchange resin method, and heating method, they all also found the protein was the main component of the EPS, followed by polysaccharide and humic acid. Among the four methods, the intensity of the two protein-like peaks of EPS extracted by heating method was the greatest, so this method can extract most completely. Moreover, this method has the least damage to cell bodies and no dissolution of intracellular substances, which was also used in this study to ensure completely and accurately extraction. 3.2.2. Infrared spectrum characteristics of bacteria EPS The results of infrared spectroscopy of EPS produced by PYR 528
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Fig. 2. Membrane potential quantified using flow cytometry in free bacteria (FB) and LBL systems (The initial PYR concentration was 50 mg L-1)
original one. Mw increased from 2.52 × 106 g mol-1 to 3.76 × 106 g mol-1 (increased 49.21%). < Rg > and values also increased from 99.68 nm to 113.45 nm–143.12 nm and 151.26 nm, respectively (the increase rates were 43.58% and 39.83%, respectively). These results indicated that the ability of microbial capture of pollutants has been improved after adding CHI. Furthermore, C* value of EPS decreased from 1.18 g L-1 to 0.72 g L-1 after the addition of CHI, indicating that the structural tightness of EPS decreases. That is to say, the addition of CHI could cause the change in the structure of EPS, including molecular chain relaxation and volume expansion. The loose structures of EPS facilitate pollutant capture and mass transfer by bacteria, thus promoting PYR removal. Similarly, Xu et al. (2013) in the study of impact by sulfadimidine added to EPS also found that sulfadimidine can make the EPS structure becomes loose, which is conducive to pollutants in the EPS mass transfer and enrichment of pollutants. In contrast, the addition of ALG in this study did not lead to obvious changes in these physical parameters, indicating that there was no obvious effect on the change in the EPS capture efficiency of contaminants.
3.2.3. Laser light scattering (LLS) characteristics of bacterial EPS In order to study whether the characteristic functional groups of EPS in bacteria CP13 may interact with the LBL microcapsule materials (CHI/ALG), thereby affect the degradation efficiency of the bacteria, the configuration changes of EPS before and after adding CHI/ALG was performed by LLS analysis. The LLS analysis can give the useful information about the EPS configuration in the solution, and the relevant physical parameters and their physical significance are as follows: < Rg > : z-average root mean square radius of gyration; : hydrodynamic radius (indicates the extent to which particles displace the surrounding solvent); C*: the inner concentration of EPS (reflects the internal density of EPS). The < Rg > and < Rh > values represent the incidence rate of interaction occurred between the compound and EPS, which are used to characterize the mass transfer process and the pollutant capture efficiency of the microorganism. The larger the value is, the stronger the ability of the microorganism to capture pollutants is. The value of C* represents the internal density of EPS, and the larger the C* value is, the closer the structure of EPS is. The configuration information of the CP13 EPS after the combination of CHI was shown in Table 1. In the presence of CHI with the final concentrations of 500 mg L-1, molecular weight (MW), < Rg > and < Rh > values of the EPS were significantly increased compared to the
3.2.4. The characteristics of the synchronous fluorescence spectra of EPS In order to investigate how CHI loses the structure of EPS, the synchronous fluorescence spectra analysis was used to study the configuration changes of EPS when combined with CHI or ALG. Results were shown in Fig. 4. It was found the peak intensity of EPS gradually 529
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Fig. 3. Cells viability quantified using flow cytometry in free bacteria (FB) and LBL systems (The initial PYR concentration was 50 mg L-1)
the tryptophan residue, which is similar to the reports of Xu et al. (2013) and Wei et al. (2015b). The two groups of researchers have demonstrated that tryptophan substances are involved in the combination of EPS and sulfamethazine (SMZ) and methylene blue (MB), respectively. On the contrary, the addition of ALG makes no obvious change of EPS fluorescence spectrum. The fluorescence intensity from the initial of 4118 down to 3668 when ALG concentrations increase from 0 μM to 400 μM (the decline was not obviously), indicating that ALG did not lead to structural changes of EPS.
decreased with the increase of CHI concentration, with intensity value decreased from 3972 to 1018 as the CHI concentration increased from 0 μM to 400 μM. The phenomena was in line with the experimental results reported by Zhang et al. (2012); Wei et al. (2015a) and Wei et al. (2016), in which fluorescent quenching for biofilm EPS bound with Cu (II); 4-chlorophenol and Zn(II) was investigated, respectively. The significant decrease of fluorescence intensity indicated that the interaction between EPS and CHI resulted in obvious fluorescence quenching and CHI had changed the structure of EPS. The fluorescence peak located at about 280nm corresponds to tryptophan-like material in protein, indicating that the decrease of fluorescence intensity is due to
Table 1 Data summary for EPS solutions in the presence and absence of CHI or ALG measured by LLS. Samples
Mw (106 g mol-1)
< Rg > (nm)
(nm)
C* (g L-1)
EPS 500 mg L-1 EPS 500 mg L-1+CHI 500 mg L-1 EPS 500 mg L-1+ALG 500 mg L-1
2.52 3.76 2.58
99.68 143.12 103.37
113.45 151.26 121.73
1.18 0.72 1.24
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Fig. 4. Synchronous fluorescence spectra of EPS with the increasing CHI (the left) or ALG (the right) concentration (EPS concentration = 100 mg L-1)
results were shown in Fig. 6 (left), from which it was found that the EEM spectra of EPS can be decomposed into two parts. They are the fluorescence peaks at excitation/emission (Ex/Em) wavelength of 220/ 342 nm and 280/342 nm, which should be ascribed to simple aromatic protein such as tyrosine (Chen et al., 2003) and aromatic amino acid tryptophan (Baker, 2001; Métivier et al., 2013; Sheng and Yu, 2006), respectively. Similar fluorescence peaks were also described in other researches (Gu et al., 2017; Li et al., 2012; Maqbool and Hur, 2016; Wang et al., 2015). The main EEM spectrum peak of EPS in this study is at wavelengths of the 220/342 nm and 280/342 nm, indicating the main component of EPS is protein. Usually fluorescence quenching can be used to indicate the change of protein structure (Xu et al., 2013), whose mechanism consists of static and dynamic quenching. In the former one, fluorescent groups and quenching agent are combined to generate stable ground state complex with no fluorescence. While in the latter one, fluorescent groups and quenching agent collide with each other and cause the disappearance of fluorescence, with no ground state compounds generated. Stem-Volmer equation is often used to analyze fluorescence quenching, the form of which is following:
3.2.5. The circular dichromatic spectrum of EPS In order to further explore the changes in the EPS structure under the action of CHI, circular dichromatic spectroscopy was used to analyze the changes of secondary structure in the protein of EPS after adding different concentrations of CHI or ALG. The results were shown in Fig. 5. It can be seen that the position of the spectral characteristic peak of the protein was shifted after different concentrations of CHI were added to the EPS. The maximum absorption wavelength moved along to the long-wave direction, from 197 nm to 204 nm, and the peak intensity also decreased with the addition of CHI. As the dosage increased from 40 μM to 80 μM, 120 μM and 200 μM, respectively, the intensity of the protein spectrum characteristic peak decreased to 22, 17, 7, and finally 5 from an original intensity of 24. The decreasing amplitude was 8.33%, 29.17%, 70.83% and 79.17%, respectively, showing an increasing tendency. Due to the fact that CHI is a non-chiral molecule, who has no circular dichromatic signal, the change of spectrum could be attributed to the change in the secondary structure of the protein in EPS, which further give evidence of the combination of CHI and EPS. This result also verified the results of the synchronous fluorescence spectra. This rule is similar to the finding of the combination of EPS and sulfamethazine (SMZ) by Xu et al. (2013). They also found that due to the invasion of the SMZ, the extension degree of the peptide chain changed, transforming the secondary structure of the protein, which contributes to the change in circular dichromatic spectrum. In this study, the intensity of the circular dichromatic spectrum peak of the EPS has no obvious change after added with different concentrations of ALG, which indicated ALG did not combine with EPS again.
F0 = 1 + K q τ0 [Q] F Where F0 and F represents the fluorescence intensity of EPS before and after the addition of the CHI or ALG, respectively; [Q] represents the concentration of CHI or ALG; Kq represents the quenching rate constant of biomolecules; τ0 is the average life span of molecules when no quenching agent is added, who has a constant value of 10-8 s (Jiang et al., 2002). 3D-EEM linear fitting results of the obtained fluorescence quenching data using the Stem-Volmer equation was showed in Fig. 6 (right). It was found that the addition of the CHI leaded to the fluorescence quenching, with the quenching rate of the protein of 1.03×1012 L mol-1
3.2.6. Analysis of EPS by 3D-EEM In order to further confirm the binding effect of CHI and EPS, 3DEEM was used to study the interaction between CHI and EPS. The
Fig. 5. Circular dichroism spectra of EPS with the increasing CHI (the left) or ALG (the right) concentration (EPS concentration = 100 mg L-1) 531
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Fig. 6. 3D-EEM fluorescence spectra of the EPS (the left) and Fitting results for fluorescence intensities of the EPS using Sterne-Volmer equation (the right) (EPS concentration = 100 mg L-1; CHI or ALG concentration = 0; 20; 40; 60; 80; 120 × 10-6 mol L-1)
s-1 (R2=0.996), which is larger than the greatest value of dynamic quenching constant measured by the interaction between any other biological molecules and all sorts of quenching agent (2.0×1010 L mol-1 s-1) (Jiang et al., 2002). This indicated that the fluorescence quenching of CHI to EPS was static fluorescence quenching, but not caused by collision (Wei et al., 2015a). Therefore, it can be speculated that CHI and EPS could form ground state complex, which suggested that CHI had been strong combined with the EPS during the encapsulation process of degrading bacteria. In order to further verify the mechanism of the EPS fluorescence quenched by CHI, the UV-visible spectrum of EPS, CHI and EPS-CHI composite were measured, and the results were shown in Fig. S1 (left). Results showed that the absorption spectrum of EPS-CHI complex did not overlap with the superimposed spectrum of EPS and CHI. The absorption at 200 nm, reflecting the framework conformation of the protein in EPS (Yang et al., 2009), decreases obviously when the CHI concentration increases, indicating that the interaction between EPS and CHI may occurred. According to the results of previous studies, if the fluorescence quenching caused by intermolecular collisions is dynamic quenching, the absorption spectrum of the mixture of EPS and the compounds is the same as that of their respective superimposed spectra. However, in this study, CHI-EPS generated from the EPS and CHI changed the absorption spectra. Therefore, the fluorescent quenching of EPS after adding of the CHI is mainly caused by the static quenching, indicating the CHI and degrading bacteria are stabilized combined with each other though the bacteria EPS. The results of UV-visible spectrum of EPS, ALG and EPS-ALG complex were shown in Fig. S2 (right), from which we can see the absorption spectrum of EPS-ALG complex was almost overlapping with the spectra of EPS, indicating that there is no stable complex formed between EPS and ALG, which further confirms the previous experimental results.
Acknowledgements This work was jointly supported by the National Key Research and Development Program of China (2018YFC1800704, 2017YFA0207003), the National Natural Science Foundation of China (41701357), the Science and Technology Planning Project of Guangdong Province, China (2016B020242004), the Maoming Public Service Platform for Transformation Upgrading and Technological Innovation of Petrochemical Industry (2016B020211001) and the Open Fund of Guangdong Provincial Key Laboratory of Petrochemical Pollution Process and Control, Guangdong University of Petrochemical Technology (No. 2018B030322017). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.06.016. References Baker, A., 2001. Fluorescence excitation-emission matrix characterization of some sewage-impacted rivers. Environ. Sci. Technol. 35, 948–953. Biswas, B., Sarkar, B., Naidu, R., 2016. Influence of thermally modified palygorskite on the viability of polycyclic aromatic hydrocarbon-degrading bacteria. Appl. Clay Sci. 134, 153–160. https://doi.org/10.1016/j.clay.2016.07.003. Caroni, A., De Lima, C.R.M., Pereira, M.R., Fonseca, J.L.C., 2012. Tetracycline adsorption on chitosan: a mechanistic description based on mass uptake and zeta potential measurements. Colloids Surfaces B Biointerfaces 100, 222–228. Chen, S., Yin, H., Ye, J., Peng, H., Liu, Z., Dang, Z., Chang, J., 2014. Influence of coexisted benzo [a] pyrene and copper on the cellular characteristics of Stenotrophomonas maltophilia during biodegradation and transformation. Bioresour. Technol. 158, 181–187. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitation− emission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37, 5701–5710. Chen, X., Ru, Y., Chen, F., Wang, X., Zhao, X., Ao, Q., 2013. FTIR spectroscopic characterization of soy proteins obtained through AOT reverse micelles. Food Hydrocolloids 31, 435–437. Clara, M., Strenn, B., Saracevic, E., Kreuzinger, N., 2004. Adsorption of bisphenol-A, 17βestradiole and 17α-ethinylestradiole to sewage sludge. Chemosphere 56, 843–851. Deng, F., Liao, C., Yang, C., Guo, C., Ma, L., Dang, Z., 2016. A new approach for pyrene bioremediation using bacteria immobilized in layer-by-layer assembled microcapsules: dynamics of soil bacterial community. RSC Adv. 6, 20654–20663. Deng, F., Zhang, Z., Yang, C., Guo, C., Lu, G., Dang, Z., 2017. Pyrene biodegradation with layer-by-layer assembly bio-microcapsules. Ecotoxicol. Environ. Saf. 138, 9–15. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, Pa, Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. El-Naas, M.H., Al-Muhtaseb, S.A., Makhlouf, S., 2009. Biodegradation of phenol by Pseudomonas putida immobilized in polyvinyl alcohol (PVA) gel. J. Hazard. Mater. 164 (2-3), 720–725. Gu, C., Gao, P., Yang, F., An, D., Munir, M., Jia, H., Xue, G., Ma, C., 2017. Characterization of extracellular polymeric substances in biofilms under long-term exposure to ciprofloxacin antibiotic using fluorescence excitation-emission matrix and parallel factor analysis. Environ. Sci. Pollut. Res. 24, 13536–13545. Günther, S., Geyer, W., Harms, H., Müller, S., 2007. Fluorogenic surrogate substrates for toluene-degrading bacteria—are they useful for activity analysis? J. Microbiol. Methods 70, 272–283. Jiang, C.-Q., Gao, M.-X., He, J.-X., 2002. Study of the interaction between terazosin and serum albumin: synchronous fluorescence determination of terazosin. Anal. Chim. Acta 452, 185–189. Khunjar, W.O., Love, N.G., 2011. Sorption of carbamazepine, 17α-ethinylestradiol,
4. Conclusions The PYR degrading bacteria CP13 immobilization of LBL microcapsules can effectively slowed down the increase of bacterial cell membrane permeability and the decrease of the membrane potential, so as to reduce the death rate and the number of cells. Component analysis results showed the component of extracellular polymers EPS of the CP13 was mainly protein, accounting for about 70%, while the other components were mainly humic acid and polysaccharide. Besides the protective effect provided by the LBL microcapsule on CP13, the other possible mechanism for LBL microcapsules promote bacterial degradation would be related to the combination of CHI and EPS of bacteria. CHI could enter the secondary structure of the protein of the EPS and combined with EPS, decreasing the density of EPS and loosening the configurations of EPS, hence improving the capture and mass transfer rate of pollutants to the bacteria, eventually promoting the bacteria degrade performance.
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1786–1795. Späth, R., Flemming, H.-C., Wuertz, S., 1998. Sorption properties of biofilms. Water Sci. Technol. 37, 207–210. Tan, L., Li, H., Ning, S., et al., 2014. Aerobic decolorization and degradation of azo dyes by suspended growing cells and immobilized cells of a newly isolated yeast Magnusiomyces ingens LH-F1. Bioresour. Technol. 158, 321–328. Verthé, K., Verstraete, W., 2006. Use of flow cytometry for analysis of phage-mediated killing of Enterobacter aerogenes. Res. Microbiol. 157, 613–618. Volkov, E.M., Nurullin, L.F., Volkov, M.E., Nikolsky, E.E., Vyskočil, F., 2011. Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+ATPase of Lumbricus terrestris body wall muscles. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 158, 520–524. Wang, L.-L., Wang, L.-F., Ren, X.-M., Ye, X.-D., Li, W.-W., Yuan, S.-J., Sun, M., Sheng, G.P., Yu, H.-Q., Wang, X.-K., 2012b. pH dependence of structure and surface properties of microbial EPS. Environ. Sci. Technol. 46, 737–744. Wang, L.-L., Wang, L.-F., Ye, X.-D., Li, W.-W., Sheng, G.-P., Yu, H.-Q., 2012a. Spatial configuration of extracellular polymeric substances of Bacillus megaterium TF10 in aqueous solution. Water Res. 46, 3490–3496. Wang, Y., Qin, J., Zhou, S., Lin, X., Ye, L., Song, C., Yan, Y., 2015. Identification of the function of extracellular polymeric substances (EPS) in denitrifying phosphorus removal sludge in the presence of copper ion. Water Res. 73, 252–264. Wei, D., Li, M., Wang, X., Han, F., Li, L., Guo, J., Ai, L., Fang, L., Liu, L., Du, B., 2016. Extracellular polymeric substances for Zn (II) binding during its sorption process onto aerobic granular sludge. J. Hazard Mater. 301, 407–415. Wei, D., Wang, B., Ngo, H.H., Guo, W., Han, F., Wang, X., Du, B., Wei, Q., 2015b. Role of extracellular polymeric substances in biosorption of dye wastewater using aerobic granular sludge. Bioresour. Technol. 185, 14–20. Wei, D., Wang, Y., Wang, X., Li, M., Han, F., Ju, L., Zhang, G., Shi, L., Li, K., Wang, B., 2015a. Toxicity assessment of 4-chlorophenol to aerobic granular sludge and its interaction with extracellular polymeric substances. J. Hazard Mater. 289, 101–107. Wu, F.J., Zhang, M.M., Guo, C.L., Liao, C.J., Chen, M.Q., Dang, Z., 2014. Effect of sources on screening and degradation ability of PAHs-degrading bacteria. Chinese Journal of Environmental Engineering 3511–3518 (in chinese). Xu, J., Sheng, G.-P., Ma, Y., Wang, L.-F., Yu, H.-Q., 2013. Roles of extracellular polymeric substances (EPS) in the migration and removal of sulfamethazine in activated sludge system. Water Res. 47, 5298–5306. Yang, Q., Liang, J., Han, H., 2009. Probing the interaction of magnetic iron oxide nanoparticles with bovine serum albumin by spectroscopic techniques. J. Phys. Chem. B 113, 10454–10458. Yang, S.-F., Lin, C.-F., Lin, A.Y.-C., Hong, P.-K.A., 2011. Sorption and biodegradation of sulfonamide antibiotics by activated sludge: experimental assessment using batch data obtained under aerobic conditions. Water Res. 45, 3389–3397. Zhang, K., Xu, Y., Hua, X., et al., 2008. An intensified degradation of phenanthrene with macroporous alginate–lignin beads immobilized Phanerochaete chrysosporium. Biochem. Eng. J. 41 (3), 251–257. Zhang, D., Lee, D.-J., Pan, X., 2012. Fluorescent quenching for biofilm extracellular polymeric substances (EPS) bound with Cu (II). J. Taiwan Inst. Chem. Eng. 43, 450–454. Zhang, Y., Liang, Y.Q., Jia, C.Y., Jiang, C.Y., Jiao, Y., 2014. Characteristics of EPS extracted from a high efficient PAHs-degrading bacterium by different methods. Chin. J. Ecol. 33 (4), 1027–1033 (in chinese). Zhang, Y.W., Prabhu, P., Lee, J.K., 2010. Alginate immobilization of recombinant Escherichia coli whole cells harboring L-arabinose isomerase for L-ribulose production. Bioproc. Biosyst. Eng. 33, 741–748.
iopromide and trimethoprim to biomass involves interactions with exocellular polymeric substances. Chemosphere 82, 917–922. Li, F., Zhu, L., 2012. Effect of surfactant-induced cell surface modifications on electron transport system and catechol 1, 2-dioxygenase activities and phenanthrene biodegradation by Citrobacter sp. SA01. Bioresour. Technol. 123, 42–48. Li, L., Gao, N., Deng, Y., Yao, J., Zhang, K., 2012. Characterization of intracellular & extracellular algae organic matters (AOM) of Microcystic aeruginosa and formation of AOM-associated disinfection byproducts and odor & taste compounds. Water Res. 46, 1233–1240. Li, W.-H., Sheng, G.-P., Liu, X.-W., Yu, H.-Q., 2008. Characterizing the extracellular and intracellular fluorescent products of activated sludge in a sequencing batch reactor. Water Res. 42, 3173–3181. Li, X.Y., Yang, S.F., 2007. Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge. Water Res. 41, 1022–1030. Lin, Q., Donghui, W., Jianlong, W., 2010. Biodegradation of pyridine by Paracoccus sp. KT-5 immobilized on bamboo-based activated carbon. Bioresour. Technol. 101 (14), 5229–5234. Lindberg, R.H., Wennberg, P., Johansson, M.I., Tysklind, M., Andersson, B.A.V., 2005. Screening of human antibiotic substances and determination of weekly mass flows in five sewage treatment plants in Sweden. Environ. Sci. Technol. 39, 3421–3429. Liu, A., Ahn, I.-S., Mansfield, C., Lion, L.W., Shuler, M.L., Ghiorse, W.C., 2001. Phenanthrene desorption from soil in the presence of bacterial extracellular polymer: observations and model predictions of dynamic behavior. Water Res. 35, 835–843. Liu, Y., Gan, L., Chen, Z., Megharaj, M., Naidu, R., 2012. Removal of nitrate using Paracoccus sp. YF1 immobilized on bamboo carbon. J. Hazard. Mater. 229, 419–425. Maqbool, T., Hur, J., 2016. Changes in fluorescent dissolved organic matter upon interaction with anionic surfactant as revealed by EEM-PARAFAC and two dimensional correlation spectroscopy. Chemosphere 161, 190–199. Métivier, R., Bourven, I., Labanowski, J., Guibaud, G., 2013. Interaction of erythromycin ethylsuccinate and acetaminophen with protein fraction of extracellular polymeric substances (EPS) from various bacterial aggregates. Environ. Sci. Pollut. Res. 20, 7275–7285. Pellicer-Nàcher, C., Domingo-Félez, C., Mutlu, A.G., Smets, B.F., 2013. Critical assessment of extracellular polymeric substances extraction methods from mixed culture biomass. Water Res. 47, 5564–5574. Sheng, G.-P., Yu, H.-Q., 2006. Characterization of extracellular polymeric substances of aerobic and anaerobic sludge using three-dimensional excitation and emission matrix fluorescence spectroscopy. Water Res. 40, 1233–1239. Sheng, G.-P., Yu, H.-Q., Li, X.-Y., 2010. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol. Adv. 28, 882–894. Sheng, G.-P., Zhang, M.-L., Yu, H.-Q., 2008. Characterization of adsorption properties of extracellular polymeric substances (EPS) extracted from sludge. Colloids Surfaces B Biointerfaces 62, 83–90. Shi, L., Günther, S., Hübschmann, T., Wick, L.Y., Harms, H., Müller, S., 2007. Limits of propidium iodide as a cell viability indicator for environmental bacteria. Cytometry 71, 592–598. Soejima, T., Iida, K., Qin, T., Taniai, H., Yoshida, S., 2009. Discrimination of live, antituberculosis agent-injured, and dead Mycobacterium tuberculosis using flow cytometry. FEMS Microbiol. Lett. 294, 74–81. Song, C., Sun, X.-F., Xing, S.-F., Xia, P.-F., Shi, Y.-J., Wang, S.-G., 2014. Characterization of the interactions between tetracycline antibiotics and microbial extracellular polymeric substances with spectroscopic approaches. Environ. Sci. Pollut. Res. 21,
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Mechanism of enhancing pyrene-degradation ability of bacteria by layer-bylayer assembly bio-microcapsules materials
T
Fucai Denga,b, Jianteng Suna, Rongni Doua,b, Wangming Denga, Yi Liua, Chen Yangb,∗, Zhi Dangb,∗ a
Guangdong Provincial Key Laboratory of Petrochemical Pollution Processes and Control, School of Environmental Science and Engineering, Guangdong University of Petrochemical Technology, Maoming, Guangdong, 525000, China b College of Environment and Energy, South China University of Technology, Guangzhou, 510006, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Layer-by-layer assembly Bio-microcapsules Flow cytometry analysis Extracellular polymeric substances Laser light scattering Fluorescence quenching
The mechanism of improving pyrene (PYR)-degrading ability of bacteria CP13 in Layer-by-layer (LBL) assembly chitosan/alginate (CHI/ALG) bio-microcapsules was investigated. Flow cytometry analysis showed that LBL microcapsules could effectively slow down the increasing rate of bacterial cell membrane permeability and the decreasing rate of the membrane potential, so as to reduce the death rate and number of the cells, which could protect the degrading bacteria. The results of Fluorescence spectrum, circular dichroism (CD) spectrum and laser light scattering (LLS) analysis revealed that the other possible mechanism for LBL microcapsules to promote bacterial degradation were following: CHI could enter the secondary structure of the protein of the extracellular polymeric substances (EPS) from CP13 and combined with EPS to generate a stable ground material, which had larger molecular weight (3.76×106 g mol-1) than the original EPS (2.52×106 g mol-1). The combination of CHI and EPS resulted in the decrease of the density of EPS from 1.18 to 0.72 g L-1, suggesting that CHI can loosen the EPS configurations, improving the capture ability of bacteria for PYR as well as the mass transfer of PYR from the extracellular to intracellular, thus eventually promoting the bacteria degrade performance.
1. Introduction Bacterial immobilization methods have been commonly employed to enhance the bioremediation of organic pollutants, including the physical adsorption of bacteria on carriers (Biswas et al., 2016; Lin et al., 2010; Liu et al., 2012) and entrapment of bacteria in alginate beads (El-Naas et al., 2009; Tan et al., 2014; Zhang et al., 2008). Compared to the free cells, immobilization has inherent advantages including enhanced stability of the system, easy separation of cells, minimizing or eliminating the cell contaminations in the products, convenient recovery and re-use of cells which enable their frequent use in the process (Zhang et al., 2010). In our previous studies, Layer-by-layer (LBL) assembly chitosan/ alginate (CHI/ALG) bio-microcapsules immobilized bacteria CP13 could gain a much higher tolerance to environmental stress (i.e. high initial pyrene (PYR) concentration, extremely low pH values or temperatures and high salt stress) (Deng et al., 2017). LBL bio-microcapsules also could cause a significant increase in the biodiversity of the bacterial community in the PYR-contaminated soil, successfully accounting for accelerated PYR removal (Deng et al., 2016). However, the mechanism was not clear yet. The strategy of bacteria immobilization
∗
to enhance PYR degradation could be attributed to the protective effect provided by the bio-microcapsule for the bacteria from the nocuity causing by the pollutant. On the other hand, the interaction which may occur between bio-microcapsules materials (CHI or ALG) and the extracellular polymeric substances (EPS) of the bacteria may be another explanation for the accelerated rate of PYR-degrading. EPS is a complex high-molecular-weight mixture of polymers produced by microorganisms, (Sheng et al., 2010). Many organic compounds, such as phenanthrene, benzene and dyes (e.g., toluidine blue), can be absorbed by EPS due to the existence of some hydrophobic regions in EPS (Liu et al., 2001; Sheng et al., 2008; Späth et al., 1998). The adsorption capacity of microbial cells decreased significantly after that EPS were removed (Khunjar and Love, 2011). Some organic polymer composite might be formed between EPS and compounds through hydrophobic interaction, hydrogen bond or electrostatic interaction (Clara et al., 2004; Lindberg et al., 2005; Yang et al., 2011), which may affect the physical and chemical properties of the EPS, resulting in the change of the degrading ability of microbial cells. However, the effect mechanism of the interaction between organic polymer composite and the EPS on the degrading ability of the cells is not clear, due to the lack of appropriate analytical tools to identify the complex interactions between them.
Corresponding authors. College of Environment and Energy, South China University of Technology, Guangzhou, China. E-mail addresses: [email protected] (F. Deng), [email protected] (C. Yang), [email protected] (Z. Dang).
https://doi.org/10.1016/j.ecoenv.2019.06.016 Received 25 February 2019; Received in revised form 24 May 2019; Accepted 5 June 2019 Available online 21 June 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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Cells, thermo fisher scientific (USA)) was used to stain the collected cells to analyze the ratio of dead, damaged and live cells of the free and LBL microencapsulated bacteria through flow cytometry, respectively. The experimental processes are as follows: firstly, the CP13 cells were collected approximately 5×106 cells mL-1. After washed 2 to 3 times with PBS, the cells was mixed with SYTO 9/PI dye solution (3 μL in proportion of 1:1) and incubated for 15 min avoiding light at room temperature. Then, the cell solution was passed through 400 mesh nylon screen, and carried out flow cytometry detection (Soejima et al., 2009). The detection excitation light was 488 nm, SYTO 9 dye was recorded on FL1 detection channel, PI dye was detected on FL3 channel, and more than 10,000 cells were counted.
In this study, the protection effects provided by the bio-microcapsules for the bacteria as well as the interactions between CHI/ALG and EPS were investigated. A combined use of fluorescence spectrum analysis, circular dichroism (CD) spectrum analysis and laser light scattering (LLS) was employed to elucidate such interaction. This study aimed to: (1) explore the protection for the cells by the LBL bio-microcapsules; (2) investigate the mechanisms for the interaction between CHI/ALG and EPS and its effect mechanism on the enhancing degrading ability of the cells. The results might favor a better understanding about the effect mechanism of cell immobilization technology on improving the cell’s degrading ability. 2. Materials and methods
2.3. EPS interacts with LBL microcapsule materials 2.1. Materials and organism 2.3.1. Extraction of EPS and its combination with CHI or ALG EPS from CP13 cells was extracted according to an improved thermal extraction method (Li and Yang, 2007). Firstly, the centrifuged cells were resuspended in 0.05% sodium chloride solution and then keep in water bath at 60 °C for 30 min. Then the bacteria solution was centrifuged for 15 min at 10000 rpm. EPS in the upper solution was collected and passed through the 0.45 μm cellulose acetate membrane. Then, EPS was further purified with 5000 Da dialysis bag to remove ions and small molecules. Finally, EPS was freeze-dried and stored in the dryer for standby. The samples for analysis the interaction between EPS and microencapsulated materials (CHI and ALG) were prepared in 50 mM (pH 7.0) PBS using 20 mL glass tube. Different volume of CHI or ALG solution was added to the tube containing 5 mL 200 mg L-1 EPS. Then, PBS was added to the mixture to make the total volume be 10 mL. Before analysis, the solution were mixed well using a vortex oscillator and balanced for 4 h.
The microorganism used in this study was Mycobacterium gilvum CP13, which was isolated from the activated sludge of a coking plant in Shaoguan, Guangdong, China (Wu et al., 2014). The bacteria were cultured in fresh nutrient broth (aqueous solution; 1.8%) and collected according to the method of Deng et al. (2017). 2.2. The protective effect of LBL microcapsules on bacteria 2.2.1. Determination of the permeability of the bacteria membrane In this study, the CP13 cells were dyed by propidium iodide (PI), and the membrane permeability of the cells are tested to figure out the characteristic changes of the CP13 cell surface. During the experiment, the CP13 cells were collected approximately 5×106 cells mL-1 during the PYR degradation process (0, 3, 5, 7d), PYR degradation using LBL bio-microcapsules was conducted in batch experiments in 100-ml shake flasks containing 20 ml of mineral salt medium (MSM) with PYR as the sole carbon source at 30°C and 180 rpm avoiding light. The MSM consisted of 2.5 g L-1 K2HPO3, 0.77 g L-1 KH2PO4, 100 mg L-1 (NH4)2SO4, 20 mg L-1 MgSO4·7H2O, 10 mg L-1 CaCl2·2H2O, 1.2 mg L-1 FeSO4·7H2O, 0.3 mg L-1 MnSO4·H2O, 0.3 mg L-1 ZnSO4·7H2O, and 0.1 mg L-1 CoSO4·7H2O, 0.1 mg L-1 (NH4)6Mo7 O24·4H2O. The initial PYR concentration was 50 mg L-1. After washing the cells for 2 to 3 times with phosphate buffer solution (PBS), they were fixed with 1% paraformaldehyde and saved under 4 °C for further analysis (Verthé and Verstraete, 2006). Before performing flow cytometry, the cells must be washed with PBS 2 to 3 times to remove the redundant paraformaldehyde. After that, enough PI solution was added to make the PI concentration as 50 μg mL-1 and the bacteria was hatched for 30 min at 37°C in the absence of lights. Finally, the bacteria solution was passed through 400 mesh nylon mesh screen, then poured into the Beckman Coulter XCL-MCL flow cytometry analysis (the test excitation light is 488 nm, FL3 detector test, 605-635 nm emission light intensity) (Günther et al., 2007).
2.3.2. Determination of bacterial EPS components (1). Polysaccharide content was analyzed using phenol-sulphate acid method (Dubois et al., 1956): 0.1 g of glucose was dissolved in 100 mL ultrapure water to obtain a standard glucose solution. 0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.4 mL standard glucose solution was added to 10 mL colorimetric tube respectively. Ultrapure water was supplemented to the volume of 1.0 mL and then 1 mL of phenol solution (5%) added and shaken well. Then 5 mL of concentrated sulfuric acid was put in and placed in a constant temperature water bath of 96°C for 20 min. When the mixture was cooled down to room temperature, the light absorbance was tested on UV spectrophotometer at the wavelength of 490 nm. The standard curve of polysaccharides was obtained with the concentration of polysaccharide as the horizontal and the absorbance as vertical coordinates, respectively. The light absorbance of 0.1 mL EPS solution (l g L-1) was determined using the same operation process. The content of polysaccharide in EPS was calculated through the standard curve. (2). The content of protein in EPS was measured by coomassie brilliant blue method with spectrophotometer under the wavelengths of 595 nm. (3). Humic acid was determined using the standard curve method and determining by UV spectrophotometer.
2.2.2. Determination of membrane potential In this study, Rhodamine 123 (Rh123) was used as the stain agent, and the cell membrane potential of free and microcapsule immobilized bacteria was quantitatively analyzed by flow cytometry during the process of PYR degradation. Rh-123 was made up to 1 mg mL-1 in ethanol and maintained in −20oC as the stain stock solution. The working concentration of Rh-123 was 10 mg mL-1 100 mL of the cell suspension was added to 400 mL of the stain in a 5 mL polypropylene tube. The mixture was incubated in a dark room at the ambient temperature for 10 min. Then, measurements were carried out using a Beckman Coulter XCL-MCL flow cytometry analysis with fluorescence detector, which detected appropriately filtered light at green (FL1, 525 nm). A total number of 10,000 cells were recorded for each sample.
The chemical structures of the EPS components were also characterized by a Fourier transform infrared (FT-IR) spectrophotometer. 2.3.3. LLS analysis LLS analysis was performed on an ALV/DLS/SLS-5022F spectrometer. Three samples (500 mg L-1 EPS, 500 mg L-1 EPS +250 mg L-1 CHI/ALG, 500 mg L-1 EPS + 500mg L-1 CHI/ALG) which prepared by 50 mm PBS (pH= 7.0) were used, and the temperature of the test was
2.2.3. Determination of the survival and damage of the bacteria SYTO 9/PI dye (LIVE-or-DIE™ Viability/Cytotoxicity Kit for Animal 526
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3.1.2. The change of cell membrane potential The changes in cell membrane potential of CP13 bacteria in free form and LBL microcapsules were shown in Fig. 2 and Table S2. It can be seen from the results that the fluorescence signal of Rh123 in the samples increased gradually, indicating that the cell membrane potential was decreased. The decrease rate of fluorescence signal in the free bacteria was faster than that of LBL microcapsules. Specifically, the fluorescence intensity of free cells decreased from the initial 141.25 to 63.10 in the 3rd day, and eventually to 3.16 in the 7th day, while the intensity value of immobilized one was still above 19 after degradation to the 7th day. Under normal circumstances, the potential of the cell membrane is negative inside and positive outside. After being stimulated, some cell membranes become positive inside and negative outside, which reduces the negative value of the membrane potential. Studies have showed that changes in cell membrane potential were closely related to changes in Na+ and K+ concentrations in- and outside the cells (Volkov et al., 2011). When cell apoptosis occurs, the integrity of the cell membrane is destroyed, the cell membrane permeability increases (Chen et al., 2014), the intracellular Na+ is gradually released into the cell, and the extracellular K+ is continuously transported to the cell via the K+-ATPase ion pump, which results in intracellular high K+ and low Na+ and extracellular environment high Na+ and low K+, eventually leading to decreased cell membrane potential. Therefore, the change of membrane permeability is the key point of cell apoptosis. In this study, the signal of Rh123 dye in the LBL microcapsule immobilized bacteria system was 6.31 times higher than the free one after 7 days of degradation, which showed that the immobilization treatment of the microcapsules slowed down the damage rate of the cell membrane integrity and the increase rate of membrane permeability. These results further confirmed that microcapsule had a protective effect on the cells, which may be the possible mechanism of the enhancement PYR degradation.
25.0 ± 0.1°C. The physical parameters of EPS were measured by dynamic light scattering and static light scattering, including the average hydraulic radius (< Rh >), z-direction mean square rotation radius (< Rg >) and apparent average molecular weight (Mw). Among them, the internal density (C*) can be calculated from Mw and < Rg > (Wang et al., 2012a). 2.3.4. Spectrum analysis The wavelength of synchronous fluorescence spectrum analysis was from 240 to 300 nm, and the excitation emission interval was 60 nm. Circular dichroism spectrum analysis was performed on a circular dichroism spectroscopy. The EPS spectra with different concentrations of CHI or ALG were recorded from 190 to 240 nm and scanned three times. Three-dimensional excitation–emission matrix (3D-EEM) fluorescence was carried out by three-dimensional fluorescence spectrophotometer (F7000, Hitachi, Japan). Excitation wavelength was 200 to 400 nm with an interval of 10 nm. Emission wavelength was 300 to 550 nm with scanning interval 0.5 nm. Ultra-pure water was scanned as a background. Specific operations refer to the literature (Li et al., 2008). EEM date is analyzed by MATLAB software. The UV absorption spectrum was analyzed by UV spectrophotometer with 1 cm cell and the wavelength range was 200 to 800 nm. 3. Results and discussion 3.1. Protective effect of LBL microcapsule on bacteria 3.1.1. The change of membrane permeability of bacteria The first step of the degradation process is the contact between microorganism and pollutants. The degradation ability of bacteria is affected at the contact step, due to the toxicity of pollutants on the bacterial cell wall and plasma membrane, which could change the microbial membrane permeability and cell membrane electric potential. It is pointed out that when the lgKow of organic solvent in contact with bacteria is 1.5–3, the fatty acid composition of bacterial cell membrane will be changed, destroying the structure and permeability of cell membrane and causing a lot of dissolution and death of bacteria, and eventually leading to the decrease of degradation rate (Shi et al., 2007). In order to explore the protective effect of LBL microcapsules for the bacteria CP13 in PYR degradation process, the changes of membrane permeability during the PYR degradation with initial concentration of 50 mg L-1 were studied in the presence and absence of microcapsules conditions. The results (Fig. 1 and Table S1) showed that during the progress of PYR degradation in both treatments, the signal of PI dye was gradually increased, indicating that the membrane permeability of the bacteria was gradually enhanced. At the initial stage, the main cells in the system were live with intact membrane structure and low permeability. The PI dye of the large molecule cannot enter the cell, and the PI signal was very weak at this stage. With the degradation time extension, the signal strength of PI detection increased, suggesting more PI molecules entered the cell and bound with DNA of bacteria. It could be concluded the integrity of the bacterial cell membrane damaged gradually, leading to the stronger permeability of cell membrane. The signal intensity of PI obtained from free bacterial treatment increased significantly faster than that of LBL immobilized bacteria, from the initial 1.41 to 22.39 on the 3rd day, and then to 50.12 on the 5th day, and finally increased to 63.10 on the 7th day. This indicates that, under the high initial concentration of PYR, the effect of PYR on the cell membrane of free bacteria leads to rapid destruction of membrane structure and strong penetration of cell membrane. After 7 days of degradation, the fluorescence intensity of the PI dye in the system was still lower than 18, which was about 1/4 of the free bacterial system, demonstrating that LBL microcapsules slowed down the damage rate of cell membrane integrity, manifesting a certain protective effect on cells.
3.1.3. The change in cell survival and damage The activity of free and LBL microcapsules immobilized CP13 cells was detected during 50 mg L-1 PYR degradation process (Fig. 3). It’s found that four groups of cells (A1, A2, A3, and A4) appeared, which were dead cells labeled by PI (red), damaged cells labeled by PI and SYTO 9 (blue), live cells labeled by SYTO 9 (green), and negative cells unlabeled (black), respectively. In the scatter plot, the changes of the proportion of the four groups of cells can directly reflect the inhibitory action of PYR on CP13 bacteria. Fig. 3 showed that excessive PYR can induce cell apoptosis during the degradation process. This may be related to the changes of functional group on cell surface, enhancing surface hydrophobicity (Li and Zhu, 2012), thereby promoting the contact of PYR with the cell surface and the migration of PYR into intracellular. When the excessive PYR enters the cell, it would produce further damage to the cell. The survival and damage of the cells treated with free and LBL microcapsule immobilized were listed in Tables S3 and S4. As can be seen from the tables, the proportion of active cells gradually decreased with the extension of the degradation time. The proportion of live cells in free bacteria decreased faster than LBL immobilized bacteria, from the 100% (initial) to 83.64% (3rd d), and then to 68.43% (5th d), and finally to 46.14% (7th d). However, the proportion of LBL immobilized bacteria in the active cells can still be remained at more than 72% at the 7th day. The results further indicated that at the high initial PYR concentration (50 mg L-1), the effect of PYR on the cell membrane of free bacteria is obvious, leading to membrane structure damage, which makes the cell death and the degradation of PYR more slowly. However, LBL immobilization treatment can make this damage effect weakened, and the rate of cell death decreased, thus make the cells showed a higher PYR degradation rate.
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Fig. 1. Cell permeability quantified using flow cytometry in free bacteria (FB) and LBL systems (The initial PYR concentration was 50 mg L-1)
degradation bacteria CP13 were shown in Fig. S1 and Table S6. The infrared spectrum exhibited a strong absorbance at 2929 cm−1, which represents C–H stretching vibrations (Caroni et al., 2012; Wang et al., 2012b). It was found that the characteristic peaks of protein in EPS appeared at 1635 and 1298 cm-1, corresponding to NH (1) (Wei et al., 2016) and amino (3) in the protein in EPS, respectively (Chen et al., 2013). The band near 1400 cm−1 was related to the absorption of C]O symmetric stretching vibration in the carboxylic groups (Chen et al., 2013; Wei et al., 2016). In addition, peaks at 1240 cm-1 can also be attributed to the C–N stretching vibration in the protein (Chen et al., 2013). Various characteristic functional groups of polysaccharide were also shown in the spectrum chart. For example, 1072 cm-1 and 1056 cm1 peaks attributed to the C–O, C–C stretching vibration (Song et al., 2014) and C–O–C, C–O–H deformation vibration of the polysacchaide, respectively, which confirms the presence of a certain amount of polysaccharide in EPS (Chen et al., 2013). In conclusion, the results of infrared characterization showed that the components of EPS produced by the CP13 bacteria have typical protein, polysaccharide characteristic spectrum (Chen et al., 2013), which verified the results of the components analysis in proportion 3.2.1.
3.2. Interaction between bacterial EPS and LBL microcapsules 3.2.1. Components analysis of the EPS The composition analysis of EPS produced by CP13 bacteria were shown in Table S5. Results showed the main component of the EPS was protein, accounting for about 70%, and followed by humic acid and polysaccharide, which were consistent with the previous studies (Gu et al., 2017; Pellicer-Nàcher et al., 2013). Similarly, when Zhang et al. (2014) extracted EPS from a PAH-degrading bacterium by four methods including cation exchange resin method, ultrasonic method, ultrasoniccation exchange resin method, and heating method, they all also found the protein was the main component of the EPS, followed by polysaccharide and humic acid. Among the four methods, the intensity of the two protein-like peaks of EPS extracted by heating method was the greatest, so this method can extract most completely. Moreover, this method has the least damage to cell bodies and no dissolution of intracellular substances, which was also used in this study to ensure completely and accurately extraction. 3.2.2. Infrared spectrum characteristics of bacteria EPS The results of infrared spectroscopy of EPS produced by PYR 528
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Fig. 2. Membrane potential quantified using flow cytometry in free bacteria (FB) and LBL systems (The initial PYR concentration was 50 mg L-1)
original one. Mw increased from 2.52 × 106 g mol-1 to 3.76 × 106 g mol-1 (increased 49.21%). < Rg > and values also increased from 99.68 nm to 113.45 nm–143.12 nm and 151.26 nm, respectively (the increase rates were 43.58% and 39.83%, respectively). These results indicated that the ability of microbial capture of pollutants has been improved after adding CHI. Furthermore, C* value of EPS decreased from 1.18 g L-1 to 0.72 g L-1 after the addition of CHI, indicating that the structural tightness of EPS decreases. That is to say, the addition of CHI could cause the change in the structure of EPS, including molecular chain relaxation and volume expansion. The loose structures of EPS facilitate pollutant capture and mass transfer by bacteria, thus promoting PYR removal. Similarly, Xu et al. (2013) in the study of impact by sulfadimidine added to EPS also found that sulfadimidine can make the EPS structure becomes loose, which is conducive to pollutants in the EPS mass transfer and enrichment of pollutants. In contrast, the addition of ALG in this study did not lead to obvious changes in these physical parameters, indicating that there was no obvious effect on the change in the EPS capture efficiency of contaminants.
3.2.3. Laser light scattering (LLS) characteristics of bacterial EPS In order to study whether the characteristic functional groups of EPS in bacteria CP13 may interact with the LBL microcapsule materials (CHI/ALG), thereby affect the degradation efficiency of the bacteria, the configuration changes of EPS before and after adding CHI/ALG was performed by LLS analysis. The LLS analysis can give the useful information about the EPS configuration in the solution, and the relevant physical parameters and their physical significance are as follows: < Rg > : z-average root mean square radius of gyration; : hydrodynamic radius (indicates the extent to which particles displace the surrounding solvent); C*: the inner concentration of EPS (reflects the internal density of EPS). The < Rg > and < Rh > values represent the incidence rate of interaction occurred between the compound and EPS, which are used to characterize the mass transfer process and the pollutant capture efficiency of the microorganism. The larger the value is, the stronger the ability of the microorganism to capture pollutants is. The value of C* represents the internal density of EPS, and the larger the C* value is, the closer the structure of EPS is. The configuration information of the CP13 EPS after the combination of CHI was shown in Table 1. In the presence of CHI with the final concentrations of 500 mg L-1, molecular weight (MW), < Rg > and < Rh > values of the EPS were significantly increased compared to the
3.2.4. The characteristics of the synchronous fluorescence spectra of EPS In order to investigate how CHI loses the structure of EPS, the synchronous fluorescence spectra analysis was used to study the configuration changes of EPS when combined with CHI or ALG. Results were shown in Fig. 4. It was found the peak intensity of EPS gradually 529
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Fig. 3. Cells viability quantified using flow cytometry in free bacteria (FB) and LBL systems (The initial PYR concentration was 50 mg L-1)
the tryptophan residue, which is similar to the reports of Xu et al. (2013) and Wei et al. (2015b). The two groups of researchers have demonstrated that tryptophan substances are involved in the combination of EPS and sulfamethazine (SMZ) and methylene blue (MB), respectively. On the contrary, the addition of ALG makes no obvious change of EPS fluorescence spectrum. The fluorescence intensity from the initial of 4118 down to 3668 when ALG concentrations increase from 0 μM to 400 μM (the decline was not obviously), indicating that ALG did not lead to structural changes of EPS.
decreased with the increase of CHI concentration, with intensity value decreased from 3972 to 1018 as the CHI concentration increased from 0 μM to 400 μM. The phenomena was in line with the experimental results reported by Zhang et al. (2012); Wei et al. (2015a) and Wei et al. (2016), in which fluorescent quenching for biofilm EPS bound with Cu (II); 4-chlorophenol and Zn(II) was investigated, respectively. The significant decrease of fluorescence intensity indicated that the interaction between EPS and CHI resulted in obvious fluorescence quenching and CHI had changed the structure of EPS. The fluorescence peak located at about 280nm corresponds to tryptophan-like material in protein, indicating that the decrease of fluorescence intensity is due to
Table 1 Data summary for EPS solutions in the presence and absence of CHI or ALG measured by LLS. Samples
Mw (106 g mol-1)
< Rg > (nm)
(nm)
C* (g L-1)
EPS 500 mg L-1 EPS 500 mg L-1+CHI 500 mg L-1 EPS 500 mg L-1+ALG 500 mg L-1
2.52 3.76 2.58
99.68 143.12 103.37
113.45 151.26 121.73
1.18 0.72 1.24
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Fig. 4. Synchronous fluorescence spectra of EPS with the increasing CHI (the left) or ALG (the right) concentration (EPS concentration = 100 mg L-1)
results were shown in Fig. 6 (left), from which it was found that the EEM spectra of EPS can be decomposed into two parts. They are the fluorescence peaks at excitation/emission (Ex/Em) wavelength of 220/ 342 nm and 280/342 nm, which should be ascribed to simple aromatic protein such as tyrosine (Chen et al., 2003) and aromatic amino acid tryptophan (Baker, 2001; Métivier et al., 2013; Sheng and Yu, 2006), respectively. Similar fluorescence peaks were also described in other researches (Gu et al., 2017; Li et al., 2012; Maqbool and Hur, 2016; Wang et al., 2015). The main EEM spectrum peak of EPS in this study is at wavelengths of the 220/342 nm and 280/342 nm, indicating the main component of EPS is protein. Usually fluorescence quenching can be used to indicate the change of protein structure (Xu et al., 2013), whose mechanism consists of static and dynamic quenching. In the former one, fluorescent groups and quenching agent are combined to generate stable ground state complex with no fluorescence. While in the latter one, fluorescent groups and quenching agent collide with each other and cause the disappearance of fluorescence, with no ground state compounds generated. Stem-Volmer equation is often used to analyze fluorescence quenching, the form of which is following:
3.2.5. The circular dichromatic spectrum of EPS In order to further explore the changes in the EPS structure under the action of CHI, circular dichromatic spectroscopy was used to analyze the changes of secondary structure in the protein of EPS after adding different concentrations of CHI or ALG. The results were shown in Fig. 5. It can be seen that the position of the spectral characteristic peak of the protein was shifted after different concentrations of CHI were added to the EPS. The maximum absorption wavelength moved along to the long-wave direction, from 197 nm to 204 nm, and the peak intensity also decreased with the addition of CHI. As the dosage increased from 40 μM to 80 μM, 120 μM and 200 μM, respectively, the intensity of the protein spectrum characteristic peak decreased to 22, 17, 7, and finally 5 from an original intensity of 24. The decreasing amplitude was 8.33%, 29.17%, 70.83% and 79.17%, respectively, showing an increasing tendency. Due to the fact that CHI is a non-chiral molecule, who has no circular dichromatic signal, the change of spectrum could be attributed to the change in the secondary structure of the protein in EPS, which further give evidence of the combination of CHI and EPS. This result also verified the results of the synchronous fluorescence spectra. This rule is similar to the finding of the combination of EPS and sulfamethazine (SMZ) by Xu et al. (2013). They also found that due to the invasion of the SMZ, the extension degree of the peptide chain changed, transforming the secondary structure of the protein, which contributes to the change in circular dichromatic spectrum. In this study, the intensity of the circular dichromatic spectrum peak of the EPS has no obvious change after added with different concentrations of ALG, which indicated ALG did not combine with EPS again.
F0 = 1 + K q τ0 [Q] F Where F0 and F represents the fluorescence intensity of EPS before and after the addition of the CHI or ALG, respectively; [Q] represents the concentration of CHI or ALG; Kq represents the quenching rate constant of biomolecules; τ0 is the average life span of molecules when no quenching agent is added, who has a constant value of 10-8 s (Jiang et al., 2002). 3D-EEM linear fitting results of the obtained fluorescence quenching data using the Stem-Volmer equation was showed in Fig. 6 (right). It was found that the addition of the CHI leaded to the fluorescence quenching, with the quenching rate of the protein of 1.03×1012 L mol-1
3.2.6. Analysis of EPS by 3D-EEM In order to further confirm the binding effect of CHI and EPS, 3DEEM was used to study the interaction between CHI and EPS. The
Fig. 5. Circular dichroism spectra of EPS with the increasing CHI (the left) or ALG (the right) concentration (EPS concentration = 100 mg L-1) 531
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Fig. 6. 3D-EEM fluorescence spectra of the EPS (the left) and Fitting results for fluorescence intensities of the EPS using Sterne-Volmer equation (the right) (EPS concentration = 100 mg L-1; CHI or ALG concentration = 0; 20; 40; 60; 80; 120 × 10-6 mol L-1)
s-1 (R2=0.996), which is larger than the greatest value of dynamic quenching constant measured by the interaction between any other biological molecules and all sorts of quenching agent (2.0×1010 L mol-1 s-1) (Jiang et al., 2002). This indicated that the fluorescence quenching of CHI to EPS was static fluorescence quenching, but not caused by collision (Wei et al., 2015a). Therefore, it can be speculated that CHI and EPS could form ground state complex, which suggested that CHI had been strong combined with the EPS during the encapsulation process of degrading bacteria. In order to further verify the mechanism of the EPS fluorescence quenched by CHI, the UV-visible spectrum of EPS, CHI and EPS-CHI composite were measured, and the results were shown in Fig. S1 (left). Results showed that the absorption spectrum of EPS-CHI complex did not overlap with the superimposed spectrum of EPS and CHI. The absorption at 200 nm, reflecting the framework conformation of the protein in EPS (Yang et al., 2009), decreases obviously when the CHI concentration increases, indicating that the interaction between EPS and CHI may occurred. According to the results of previous studies, if the fluorescence quenching caused by intermolecular collisions is dynamic quenching, the absorption spectrum of the mixture of EPS and the compounds is the same as that of their respective superimposed spectra. However, in this study, CHI-EPS generated from the EPS and CHI changed the absorption spectra. Therefore, the fluorescent quenching of EPS after adding of the CHI is mainly caused by the static quenching, indicating the CHI and degrading bacteria are stabilized combined with each other though the bacteria EPS. The results of UV-visible spectrum of EPS, ALG and EPS-ALG complex were shown in Fig. S2 (right), from which we can see the absorption spectrum of EPS-ALG complex was almost overlapping with the spectra of EPS, indicating that there is no stable complex formed between EPS and ALG, which further confirms the previous experimental results.
Acknowledgements This work was jointly supported by the National Key Research and Development Program of China (2018YFC1800704, 2017YFA0207003), the National Natural Science Foundation of China (41701357), the Science and Technology Planning Project of Guangdong Province, China (2016B020242004), the Maoming Public Service Platform for Transformation Upgrading and Technological Innovation of Petrochemical Industry (2016B020211001) and the Open Fund of Guangdong Provincial Key Laboratory of Petrochemical Pollution Process and Control, Guangdong University of Petrochemical Technology (No. 2018B030322017). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.06.016. References Baker, A., 2001. Fluorescence excitation-emission matrix characterization of some sewage-impacted rivers. Environ. Sci. Technol. 35, 948–953. Biswas, B., Sarkar, B., Naidu, R., 2016. Influence of thermally modified palygorskite on the viability of polycyclic aromatic hydrocarbon-degrading bacteria. Appl. Clay Sci. 134, 153–160. https://doi.org/10.1016/j.clay.2016.07.003. Caroni, A., De Lima, C.R.M., Pereira, M.R., Fonseca, J.L.C., 2012. Tetracycline adsorption on chitosan: a mechanistic description based on mass uptake and zeta potential measurements. Colloids Surfaces B Biointerfaces 100, 222–228. Chen, S., Yin, H., Ye, J., Peng, H., Liu, Z., Dang, Z., Chang, J., 2014. Influence of coexisted benzo [a] pyrene and copper on the cellular characteristics of Stenotrophomonas maltophilia during biodegradation and transformation. Bioresour. Technol. 158, 181–187. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitation− emission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37, 5701–5710. Chen, X., Ru, Y., Chen, F., Wang, X., Zhao, X., Ao, Q., 2013. FTIR spectroscopic characterization of soy proteins obtained through AOT reverse micelles. Food Hydrocolloids 31, 435–437. Clara, M., Strenn, B., Saracevic, E., Kreuzinger, N., 2004. Adsorption of bisphenol-A, 17βestradiole and 17α-ethinylestradiole to sewage sludge. Chemosphere 56, 843–851. Deng, F., Liao, C., Yang, C., Guo, C., Ma, L., Dang, Z., 2016. A new approach for pyrene bioremediation using bacteria immobilized in layer-by-layer assembled microcapsules: dynamics of soil bacterial community. RSC Adv. 6, 20654–20663. Deng, F., Zhang, Z., Yang, C., Guo, C., Lu, G., Dang, Z., 2017. Pyrene biodegradation with layer-by-layer assembly bio-microcapsules. Ecotoxicol. Environ. Saf. 138, 9–15. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, Pa, Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. El-Naas, M.H., Al-Muhtaseb, S.A., Makhlouf, S., 2009. Biodegradation of phenol by Pseudomonas putida immobilized in polyvinyl alcohol (PVA) gel. J. Hazard. Mater. 164 (2-3), 720–725. Gu, C., Gao, P., Yang, F., An, D., Munir, M., Jia, H., Xue, G., Ma, C., 2017. Characterization of extracellular polymeric substances in biofilms under long-term exposure to ciprofloxacin antibiotic using fluorescence excitation-emission matrix and parallel factor analysis. Environ. Sci. Pollut. Res. 24, 13536–13545. Günther, S., Geyer, W., Harms, H., Müller, S., 2007. Fluorogenic surrogate substrates for toluene-degrading bacteria—are they useful for activity analysis? J. Microbiol. Methods 70, 272–283. Jiang, C.-Q., Gao, M.-X., He, J.-X., 2002. Study of the interaction between terazosin and serum albumin: synchronous fluorescence determination of terazosin. Anal. Chim. Acta 452, 185–189. Khunjar, W.O., Love, N.G., 2011. Sorption of carbamazepine, 17α-ethinylestradiol,
4. Conclusions The PYR degrading bacteria CP13 immobilization of LBL microcapsules can effectively slowed down the increase of bacterial cell membrane permeability and the decrease of the membrane potential, so as to reduce the death rate and the number of cells. Component analysis results showed the component of extracellular polymers EPS of the CP13 was mainly protein, accounting for about 70%, while the other components were mainly humic acid and polysaccharide. Besides the protective effect provided by the LBL microcapsule on CP13, the other possible mechanism for LBL microcapsules promote bacterial degradation would be related to the combination of CHI and EPS of bacteria. CHI could enter the secondary structure of the protein of the EPS and combined with EPS, decreasing the density of EPS and loosening the configurations of EPS, hence improving the capture and mass transfer rate of pollutants to the bacteria, eventually promoting the bacteria degrade performance.
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F. Deng, et al.
1786–1795. Späth, R., Flemming, H.-C., Wuertz, S., 1998. Sorption properties of biofilms. Water Sci. Technol. 37, 207–210. Tan, L., Li, H., Ning, S., et al., 2014. Aerobic decolorization and degradation of azo dyes by suspended growing cells and immobilized cells of a newly isolated yeast Magnusiomyces ingens LH-F1. Bioresour. Technol. 158, 321–328. Verthé, K., Verstraete, W., 2006. Use of flow cytometry for analysis of phage-mediated killing of Enterobacter aerogenes. Res. Microbiol. 157, 613–618. Volkov, E.M., Nurullin, L.F., Volkov, M.E., Nikolsky, E.E., Vyskočil, F., 2011. Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+ATPase of Lumbricus terrestris body wall muscles. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 158, 520–524. Wang, L.-L., Wang, L.-F., Ren, X.-M., Ye, X.-D., Li, W.-W., Yuan, S.-J., Sun, M., Sheng, G.P., Yu, H.-Q., Wang, X.-K., 2012b. pH dependence of structure and surface properties of microbial EPS. Environ. Sci. Technol. 46, 737–744. Wang, L.-L., Wang, L.-F., Ye, X.-D., Li, W.-W., Sheng, G.-P., Yu, H.-Q., 2012a. Spatial configuration of extracellular polymeric substances of Bacillus megaterium TF10 in aqueous solution. Water Res. 46, 3490–3496. Wang, Y., Qin, J., Zhou, S., Lin, X., Ye, L., Song, C., Yan, Y., 2015. Identification of the function of extracellular polymeric substances (EPS) in denitrifying phosphorus removal sludge in the presence of copper ion. Water Res. 73, 252–264. Wei, D., Li, M., Wang, X., Han, F., Li, L., Guo, J., Ai, L., Fang, L., Liu, L., Du, B., 2016. Extracellular polymeric substances for Zn (II) binding during its sorption process onto aerobic granular sludge. J. Hazard Mater. 301, 407–415. Wei, D., Wang, B., Ngo, H.H., Guo, W., Han, F., Wang, X., Du, B., Wei, Q., 2015b. Role of extracellular polymeric substances in biosorption of dye wastewater using aerobic granular sludge. Bioresour. Technol. 185, 14–20. Wei, D., Wang, Y., Wang, X., Li, M., Han, F., Ju, L., Zhang, G., Shi, L., Li, K., Wang, B., 2015a. Toxicity assessment of 4-chlorophenol to aerobic granular sludge and its interaction with extracellular polymeric substances. J. Hazard Mater. 289, 101–107. Wu, F.J., Zhang, M.M., Guo, C.L., Liao, C.J., Chen, M.Q., Dang, Z., 2014. Effect of sources on screening and degradation ability of PAHs-degrading bacteria. Chinese Journal of Environmental Engineering 3511–3518 (in chinese). Xu, J., Sheng, G.-P., Ma, Y., Wang, L.-F., Yu, H.-Q., 2013. Roles of extracellular polymeric substances (EPS) in the migration and removal of sulfamethazine in activated sludge system. Water Res. 47, 5298–5306. Yang, Q., Liang, J., Han, H., 2009. Probing the interaction of magnetic iron oxide nanoparticles with bovine serum albumin by spectroscopic techniques. J. Phys. Chem. B 113, 10454–10458. Yang, S.-F., Lin, C.-F., Lin, A.Y.-C., Hong, P.-K.A., 2011. Sorption and biodegradation of sulfonamide antibiotics by activated sludge: experimental assessment using batch data obtained under aerobic conditions. Water Res. 45, 3389–3397. Zhang, K., Xu, Y., Hua, X., et al., 2008. An intensified degradation of phenanthrene with macroporous alginate–lignin beads immobilized Phanerochaete chrysosporium. Biochem. Eng. J. 41 (3), 251–257. Zhang, D., Lee, D.-J., Pan, X., 2012. Fluorescent quenching for biofilm extracellular polymeric substances (EPS) bound with Cu (II). J. Taiwan Inst. Chem. Eng. 43, 450–454. Zhang, Y., Liang, Y.Q., Jia, C.Y., Jiang, C.Y., Jiao, Y., 2014. Characteristics of EPS extracted from a high efficient PAHs-degrading bacterium by different methods. Chin. J. Ecol. 33 (4), 1027–1033 (in chinese). Zhang, Y.W., Prabhu, P., Lee, J.K., 2010. Alginate immobilization of recombinant Escherichia coli whole cells harboring L-arabinose isomerase for L-ribulose production. Bioproc. Biosyst. Eng. 33, 741–748.
iopromide and trimethoprim to biomass involves interactions with exocellular polymeric substances. Chemosphere 82, 917–922. Li, F., Zhu, L., 2012. Effect of surfactant-induced cell surface modifications on electron transport system and catechol 1, 2-dioxygenase activities and phenanthrene biodegradation by Citrobacter sp. SA01. Bioresour. Technol. 123, 42–48. Li, L., Gao, N., Deng, Y., Yao, J., Zhang, K., 2012. Characterization of intracellular & extracellular algae organic matters (AOM) of Microcystic aeruginosa and formation of AOM-associated disinfection byproducts and odor & taste compounds. Water Res. 46, 1233–1240. Li, W.-H., Sheng, G.-P., Liu, X.-W., Yu, H.-Q., 2008. Characterizing the extracellular and intracellular fluorescent products of activated sludge in a sequencing batch reactor. Water Res. 42, 3173–3181. Li, X.Y., Yang, S.F., 2007. Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge. Water Res. 41, 1022–1030. Lin, Q., Donghui, W., Jianlong, W., 2010. Biodegradation of pyridine by Paracoccus sp. KT-5 immobilized on bamboo-based activated carbon. Bioresour. Technol. 101 (14), 5229–5234. Lindberg, R.H., Wennberg, P., Johansson, M.I., Tysklind, M., Andersson, B.A.V., 2005. Screening of human antibiotic substances and determination of weekly mass flows in five sewage treatment plants in Sweden. Environ. Sci. Technol. 39, 3421–3429. Liu, A., Ahn, I.-S., Mansfield, C., Lion, L.W., Shuler, M.L., Ghiorse, W.C., 2001. Phenanthrene desorption from soil in the presence of bacterial extracellular polymer: observations and model predictions of dynamic behavior. Water Res. 35, 835–843. Liu, Y., Gan, L., Chen, Z., Megharaj, M., Naidu, R., 2012. Removal of nitrate using Paracoccus sp. YF1 immobilized on bamboo carbon. J. Hazard. Mater. 229, 419–425. Maqbool, T., Hur, J., 2016. Changes in fluorescent dissolved organic matter upon interaction with anionic surfactant as revealed by EEM-PARAFAC and two dimensional correlation spectroscopy. Chemosphere 161, 190–199. Métivier, R., Bourven, I., Labanowski, J., Guibaud, G., 2013. Interaction of erythromycin ethylsuccinate and acetaminophen with protein fraction of extracellular polymeric substances (EPS) from various bacterial aggregates. Environ. Sci. Pollut. Res. 20, 7275–7285. Pellicer-Nàcher, C., Domingo-Félez, C., Mutlu, A.G., Smets, B.F., 2013. Critical assessment of extracellular polymeric substances extraction methods from mixed culture biomass. Water Res. 47, 5564–5574. Sheng, G.-P., Yu, H.-Q., 2006. Characterization of extracellular polymeric substances of aerobic and anaerobic sludge using three-dimensional excitation and emission matrix fluorescence spectroscopy. Water Res. 40, 1233–1239. Sheng, G.-P., Yu, H.-Q., Li, X.-Y., 2010. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol. Adv. 28, 882–894. Sheng, G.-P., Zhang, M.-L., Yu, H.-Q., 2008. Characterization of adsorption properties of extracellular polymeric substances (EPS) extracted from sludge. Colloids Surfaces B Biointerfaces 62, 83–90. Shi, L., Günther, S., Hübschmann, T., Wick, L.Y., Harms, H., Müller, S., 2007. Limits of propidium iodide as a cell viability indicator for environmental bacteria. Cytometry 71, 592–598. Soejima, T., Iida, K., Qin, T., Taniai, H., Yoshida, S., 2009. Discrimination of live, antituberculosis agent-injured, and dead Mycobacterium tuberculosis using flow cytometry. FEMS Microbiol. Lett. 294, 74–81. Song, C., Sun, X.-F., Xing, S.-F., Xia, P.-F., Shi, Y.-J., Wang, S.-G., 2014. Characterization of the interactions between tetracycline antibiotics and microbial extracellular polymeric substances with spectroscopic approaches. Environ. Sci. Pollut. Res. 21,
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