Bioresource Technology 158 (2014) 181–187

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Influence of co-existed benzo[a]pyrene and copper on the cellular characteristics of Stenotrophomonas maltophilia during biodegradation and transformation Shuona Chen a, Hua Yin b,⇑, Jinshao Ye c, Hui Peng d, Zehua Liu b, Zhi Dang b, Jingjing Chang c a

Department of Ecology, Jinan University, Guangzhou 510632, China Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, College of Environment and Energy, South China University of Technology, Guangzhou 510006, China Department of Environmental Engineering, Jinan University, Guangzhou 510632, China d Department of Chemistry, Jinan University, Guangzhou 510632, China b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 BaP was stored in microbial cell first

instead of being decomposed instantly.  The presence of BaP and Cu(II) strengthened the microbial membrane permeability.  Higher esterase activity and DNA in microbial cells still existed after reaction.  FCM and TEM were used to study the mechanism of bio-remediation on a cell level.

a r t i c l e

i n f o

Article history: Received 18 November 2013 Received in revised form 3 February 2014 Accepted 7 February 2014 Available online 17 February 2014 Keywords: Cellular characteristics Benzo[a]pyrene Copper Stenotrophomonas maltophilia Flow cytometry

a b s t r a c t Microbial remediation has been proposed as a promising technique to remove pollutions, however, its application has been hindered by the lack of understanding the mechanisms involved in contaminants conversion and the influence of pollutants on cellular characteristics. To address this problem, biodegradation and transformation of BaP–Cu(II) by Stenotrophomonas maltophilia, along with interactions of these pollutants with microbial cells through FCM assay were investigated. The results indicated that BaP and Cu(II) were rapidly removed by S. maltophilia on the 1st d, but only less than 10% BaP was broken down due to temporary store in cells, instead of being decomposed immediately. The key ATP enzymes in cells were then activated by BaP to promote bacteria to further decompose BaP. Stimulation of co-existed contaminants strengthened cell membrane permeability and altered cell structure, but a higher esterase activity and DNA in cells of S. maltophilia were still retained. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Benzo[a]pyrene (BaP) and copper (Cu) are ubiquitous pollutants usually coexisting at contaminated sites. They are prone to binding

⇑ Corresponding author. Tel.: +86 20 39380569. E-mail address: [email protected] (H. Yin). http://dx.doi.org/10.1016/j.biortech.2014.02.020 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

onto sediments and particles in water and soils, which might accumulate through food chains, leading to great harm on human health and environment (Fu et al., 2013; Liu et al., 2012; Rossi et al., 2012). Although chemical oxidation or photolysis might affect the fate of BaP and Cu, microbial degradation and transformation have been reported as the more effective approaches for removing BaP and Cu from environment (Lei et al., 2005; Tandy et al., 2004; Xu et al., 2013).

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Cell wall and plasma membrane can make microbe avoid the toxic effects of increased permeability induced by hazardous substances including organic compounds and heavy metals (Chakravarty and Banerjee, 2012; Song et al., 2009; Wang et al., 2003), but they are also the first barrier for microbe to decompose pollutants. After being in touch with bacteria, contaminants would first adhere to the cell surface, and then enter into cells by means of ion channels (Kishore et al., 2011), or by a transmembrane carrier (Gokel George and Daschbach, 2007), or through self-diffusion owing to ‘‘holes’’ effect on cell film. Next, contaminants would be decomposed or transformed under the action of intracellular proteases (Naoyuki et al., 2004). Yonezawa et al. reported that the mechanism of organic matter biodegradation was mainly due to intracellular enzymes function which included adsorption, transmembrane transport, and enzymatic reaction (Susarla et al., 1998; Yan et al., 2009). To date, however, there is still uncertain gray area about the microscopic mechanism of organic matter biodegradation and heavy metal biosorption. In particular, the influence of organic compounds and heavy metals on microbial properties is more complex when these two pollutants co-exist. Our previous study has confirmed that the cell surface hydrophobicity and membrane permeability of Stenotrophomonas maltophilia were all affected during the bioremoval of BaP–Cu(II) combined pollutants (Chen et al., 2012). Fang et al. (2000) compared physiological effect of organic compounds such as benzene and toluene etc. on five different kinds of microorganisms, and analyzed the changes of cell membrane simultaneously, by using liquid chromatography/spray ionization/mass spectrometry (LC/ESI/MS). The research conclusions drawn by scholars (Chi et al., 2006; Molatová et al., 2010; Mark et al., 2000) were that the toxicity of organic compounds and heavy metals influenced permeability of microbial membrane, which pertained to the repair capacity of microorganisms. And this toxic level was related to the types of pollutants, as well as nature of microbial cell. The interactions between microorganisms and contaminants will alter the characteristics of cell surface, and affect the cell activity and ion metabolism as well, which is bound to influence uptake and removal of pollutants. Therefore, the emphasis of research on degradation performance has shifted from a macro perspective to the micro mechanism of pollutants uptake, as well as the effect of pollutants on microbial membrane feature and cell activity, which will be beneficial to expound the limiting factors during microbial remediation of environmental pollution, so as to design targeted measures to significantly improve removal efficiency. In this research we chose BaP and Cu(II) as representative materials of PAHs and heavy metals, to study the removal of BaP and Cu(II) combined pollutants using S. maltophilia. The investigation primarily focused on the changes of cell activity, cell structure and membrane permeability caused by pollutants by means of flow cytometry (FCM), aiming to more thoroughly reveal the mechanism of combined pollutants uptake in microbial remediation.

2. Methods 2.1. Strain, medium and chemicals 2.1.1. Strain Strain used in this study was Stenotrophomonas maltophilia (S. maltophilia), a potential strain for BaP biodegradation and Cu(II) biosorption, isolated from an e-waste dismantling area in Guiyu town of Guangdong province, China, and was determined as gram-negative.

2.1.2. Medium and chemicals Two kinds of culture mediums were used. Enrichment medium was used for strain culture, and its composition was as follows (g L1): beef extracts (3), peptone (10), NaCl (5). Mineral salt medium (MSM) was used as the degradation medium, and its composition was as follows (g L1): (NH4)2SO4 (1), K2HPO4 (7), KH2PO4 (3), MgSO47H2O (0.1), sodium citrate (0.5), pH was from 7.0 to 7.2. All the mediums were previously sterilized in an autoclave at 121 °C for 30 min. BaP was obtained from Sigma–Aldrich (St. Louis, MO, USA). A stock solution (1000 mg L1) of Cu(II) ions was prepared by dissolving Cu(NO3)2 (AR grade) in distilled water and sterilized by passing through Millipore membrane filters (0.22 mm). The stock solution was then appropriately diluted to obtain the test solutions of desired strength. 2.2. Biodegradation and biosorption of BaP and Cu(II) All biodegradation experiments were carried out in batch by shaking a Erlenmeyer flask containing 20 mL MSM solution with BaP (1 mg L1), Cu(II) ions (10 mg L1), and BaP (1 mg L1)-Cu(II) ions (10 mg L1) respectively, and consortium at a concentration of 2.5 g L1 on a rotary shaker at 130 r min1 in the dark for the desired time. Sample without consortium was served as control. Subsequently, samples of individual flasks were completely collected at the desired time respectively, with the cell suspensions being broken by ultrasonic pulverizer to estimate intracellular BaP and Cu(II) concentration, and the solution to measure the residual content of extracellular BaP and Cu(II). 2.3. Analytical method of BaP and Cu(II) The residual BaP in samples was extracted by dichloromethane and analyzed by high-performance liquid chromatography (HPLC) with UV detector at 254 nm and C18 reverse-phase column (dimensions 0.25 mm  150 mm), methanol–water (95/5, v/v) was used with a flow rate of 1 mL min1 as a mobile phase. The injection volume was 20 lL (Chen et al., 2013). Removal and degradation rate (%) of BaP are calculated as follows:

Qr ¼

ðC 0  C t Þ  100 C0

ð1Þ

where, Qr represents removal ratios of BaP(%) in a certain period of time, C0 and Ct are initial and final BaP concentration in the solution, individually.

Qd ¼

C 0  ðC i þ C e Þ  100 C0

ð2Þ

where, Qd stands for degradation ratios of BaP(%) in a certain period of time, C0 , Ci and Ce are initial, intracellular and extracellular BaP concentration separately. The residual concentrations of Cu after biosorption were determined by AA-7000 atomic absorption spectrophotometer (Shimadzu, Japan). The removal ratios of Cu and biosorption capacity are calculated as follows:

Q 0r ¼

ðC 00  C 0t Þ  100 C 00

ð3Þ

where Q0 r represents removal ratio of Cu(%), C0 0 and C0 t are initial and final Cu concentration, respectively.

QC ¼

ðC 00  C 0t Þ  100 Cb

ð4Þ

where Qc stands for biosorption capacity (mg g1), C0 0 and C0 t are initial and final Cu concentration, individually, and Cb is biomass concentration.

S. Chen et al. / Bioresource Technology 158 (2014) 181–187

All of the experiments were performed in triplicate, and the mean values were used in the calculations.

2.4. Flow cytometric analysis 2.4.1. Sample preparation After reactions between S. maltophilia and BaP/Cu(II), bacterial cells were harvested and washed twice with high purity water, then re-suspended in sterile 0.1 M phosphate buffer (PBS, pH 7.0), and the optical density was set at defined values used for staining tests with a combination of Propidium Iodide (PI, Sigma–Aldrich, Dublin, Ireland) and Fluorescein diacetat (FDA, Sigma–Aldrich, Dublin, Ireland).

2.4.2. Staining Thallus was firstly stained with FDA to a final concentration of 0.005 mg mL1. After a thorough mixing, samples were placed in the dark at 37 °C for 15 min, then analyzed using FCM to measure the permeability of cell membrane and esterase activity. Thereafter, samples were stained with a final concentration of 0.05 mg mL1 PI, mixed, and left in the dark at room temperature for 10 min (Antje et al., 2012; Daniel et al., 2003). After removing excessive dye, the cell pellet was re-suspended in 0.1 M PBS to obtain a cell concentration of approximately 106 bacteria cells per milliliter.

2.4.3. FCM analysis FCM analysis was performed on a FACSAria flow cytometer (BD, USA). The green fluorescence (from FDA) and red fluorescence (from PI) were captured through a 530/30 nm band-pass filter and a 670 nm long-pass filter, respectively. Samples were acquired using a flow rate of 10 lL min1, with 10,000 events being acquired per sample. The software used for data acquisition and analysis was FACSDiva (BD, USA).

2.5. Cell disruption and ATPase activity test Bacterial cells in the reaction system were collected and washed twice, suspended in 1 mL distilled water. A needle probe with 2 mm diameter was used at 20 kHz in the experiment (JY92-II, Scieniz Inc., China). Cell disruption was carried out at 450 W for 20 min (5 s: 9 s pulse on: off basis). After then, the cell wall and cell debris were separated from the suspension by centrifuge (KDC160HR, Zonkia Scientific Instruments Co., Ltd., China) at 6000 r min1 for 10 min at 4 °C. The cell inclusion was obtained and used for ATPase activity assay that was run strictly according to the instruction of ATPase activity test kit which was provided by Nanjing Jiancheng Bioengineering Institute, China.

2.6. Protein concentration estimation Total cell protein was estimated using the method of Bradford (Knight and Chambers, 2003). All samples were allowed to stand for 5 min, and absorbance was recorded at 595 nm.

2.7. Intracellular morphology of S. maltophilia The control samples and bacterial cells in reaction systems were fixed and dehydrated firstly. Then, the samples were embedded in an epoxy resin and cured at 50 °C for 72 h. After then, the samples were cut into ultra-thin slices for further transmission electron microscopy (TEM, Philips Tecnai 10) observation.

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3. Results and discussion 3.1. Relationship between BaP biodegradation/Cu(II) biosorption and reaction time The ability of S. maltophilia to decompose BaP and remove Cu(II) in the pollution systems of BaP, BaP–Cu(II) and Cu(II) during 1–12 d was investigated and the result was presented in Fig. 1. The experiment in Fig. 1-(1) revealed that the removal ratio and degradation efficiency of 1 mg L1 BaP by S. maltophilia were increasing with time. BaP removal was enhanced rapidly and reached to 67.1% on the 1st d, but was slightly inhibited and came to 56.9% when there was Cu(II) in the solution. During this time, however, only a small amount of BaP was broken down with degradation ratio of less than 10%, instead, most of BaP was transferred to the cell surface or inside of bacteria. Two days later, the removal efficiency of BaP further increased in different pollution systems, and reached to 76.9% (BaP system) and 68.5% (BaP–Cu(II) system) on the 4th d. After then, the removal tended to stabilize. However, it was illustrated from Fig. 1-(1) that BaP removal ratio was obviously higher than its degradation ratio. BaP transferred from solution was not decomposed quickly and fully utilized by S. maltophilia. From the 1st d on, the BaP degradation efficiency increased sharply and reached 46.6% after 4 d, but was still lower than the removal ratio of 76.9% over the same period. Then, the degradation ratio increased slowly with prolonging time. Compared with single BaP pollution system, BaP decomposition slowed down when there were 10 mg L1 Cu(II) ions in the solutions, and this decomposition received a rapid ascending on the 5th d, then went to the highest point of 54.5% on the 7th d. The experimental data showed that only 60% of the removed BaP was decomposed and finally used due to the action of S. maltophilia, although most of BaP had been removed from the solution. There was still a small part of BaP accumulating inside the bacterial cells, which was detected from 0.06 to 0.12 mg g1 after 5 d. In this study we also determined the transport of intracellular and extracellular Cu(II) in order to further verify the interaction between bacteria and heavy metals. The results were depicted in Fig. 1-(2). It can be found that the Cu(II) migration in different pollution systems changed regularly over time. From the 4th h on, the concentration of Cu(II) in extracellular solution declined rapidly both in the single Cu(II) ions and BaP–Cu(II) ions combined systems, while the Cu(II) content inside cells continually increased. Then it maintained balance on the 2nd d, with Cu(II) concentration of outside and inside bacterial cells kept in 2.4–3.6 mg L1 and 1.2–1.5 mg g1, respectively. Nevertheless, the intracellular Cu(II) ions began to release slowly after 7 d, and stabilized at 0.3–0.6 mg g1 finally. Consequently, it can be concluded from this experiment that S. maltophilia activated the key intracellular protease after adapting to contaminants for a short time, and began to break down BaP and utilize it as energy to promote their own growth and metabolism. Unlike BaP degradation, most of Cu(II) were adsorbed on cell surface with some passing through the membrane into cells.

3.2. Effect of pollutants on bacterial cells The information obtained by FCM is mainly from specific and non-specific fluorescence signal. Scattered light intensity and spatial distribution is closely related to the size, shape, membrane and internal structure of cells, and the cells that have not suffered any damage possess the characteristic of light scattering. There are two common kinds of scattered light to be used during FCM analysis. One is forward scattering (FSC), which is associated with the cell size, and will increase with the cell cross-sectional area for the

S. Chen et al. / Bioresource Technology 158 (2014) 181–187

70

60

60

50

50

40

40

30

30 remvval,BaP system removal,BaP-Cu system degradation,BaP system degradation,BaP-Cu system

20 10 0

20

10

10 0

0

2

4

6 time/d

8

10

12 BaP-Cu Extracellular Cu Extracellular BaP-Cu Intracellular Cu Intracellular

12

(1)

8

10 -1

70

12

8

6

6

4

4

2

2

0

Cu content/mg.g

80 -1

90

80

Cu concentration/mg.L

90

BaP degradation rate/%

BaP removal rate/ %

184

0 0

2

4

6 time/d

8

10

12

(2)

Fig. 1. Change of BaP degradation and Cu(II) biosorption by S. maltophilia with time. (1) Removal and degradation efficiency of BaP with increasing time, in single BaP (1 mg L1) and BaP (1 mg L1)-Cu(II) (10 mg L1) combined systems. Reactions were taken place at 30 °C with shaking at 130 r min1. (2) Change of Cu(II) transport with increasing time, in single Cu(II) (10 mg L1) and BaP (1 mg L1)–Cu(II) (10 mg L1) combined system. Reactions were taken place at 30 °C with shaking at 130 r min1.

same cell. The other is side scattering (SSC), which is mainly used to obtain information related to the nature of the internal granule cells. Usually the larger the particle density inside the cell is, the greater the SSC value is (Wang et al., 2010). In this study, we used FCM to analyze the bacterial community in pollution systems after 5, 7 and 12 d of decomposing and removing contaminants by S. maltophilia, and the results were presented in Fig. 2. It can be seen from Fig. 2-(a–d) that the bacterial cells in the control and single BaP pollution systems were relatively concentrated. When there was Cu(II) in the solution, the bacterial cells scattering deviated after 5 d, with its SSC value becoming smaller, and FSC value turning to larger. Especially when bacteria interacted with Cu(II) for 12 d (Fig. 2-d-3), a group of dispersed particles were detected below the aggregated cells. This result showed that varying degrees of changes occurred in cell size and intracellular particle density under the stimulation of different pollutants, in

particular, Cu(II) ions, which contacted with the bacterial cells and generated interaction. As a result, cell wall and membrane were ruptured, which led to change of cell osmotic pressure and subsequent entering of extracellular substances, and finally gave rise to swell of bacterial cell. Besides, FSC value increased due to the adsorption of BaP and Cu(II) ions on the surface of bacterial cell. In addition, since ‘‘holes’’ appeared on the cell surface resulted in cytoplasm outflow, some cells had only empty shell left, which made the value of cell populations SSC shift downward. In comparison with Fig. 2-(1–3) in the same system, it was found that the cell population in the control group had no obvious changes and still remained relatively concentrated over time. The similar phenomena appeared when there was only BaP in the system, suggesting that the cell size and shape were not seriously affected although the internal structure of bacterial cells slightly changed after 12 d exposure with cytoplasm particle density decreased. When BaP and Cu(II) co-existed in the solution, however, significant deviation

Fig. 2. Influence of BaP and Cu(II) on the characteristics of S. maltophilia cell a-1 to a-3 stands for the changes of size and density of cells in control system after 5, 7 and 12 d, separately. b-1 to b-3, c-1 to c-3, d-1 to d-3 depict the changes of size and density of cells that have interacted with BaP (1 mg L1), BaP (1 mg L1)–Cu(II) (10 mg L1), and Cu(II) (10 mg L1), respectively for 5, 7 and 12 d. In the figures, the abscissa represents the relative intensity of FSC, and the ordinate stands for that of SSC.

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on the bacterial cells appeared on the 5th d. This implied that heavy metal Cu(II) made greater effect on S. maltophilia cells. It can be found from Fig. 1-(2) that at the beginning of bioremediation, Cu(II) ions were quickly transported into the cells and interacted with the intracellular proteins. As a result, some of Cu(II) ions were chelated to form macromolecular compounds, such as metallothionein, through which the amount of free copper ions that had greater destructive effect was reduced, consequently protecting more important biological molecules. As cell membrane permeability of S. maltophilia increased, the intracellular copper chelate gradually flowed out, leading to the ascended Cu(II) content outside cells. At the same time, it was detected that the SSC value of cells went down, which represented the decreased inclusion density. Even on the 12th d, some small particles that were probably copper chelate or microbial cell debris, dispersed in the solution were still increasing significantly (shown in Fig. 2-d-3). 3.3. Analysis of activity and integrity of bacterial cells in different growth phase Non-permeant dye PI cannot enter the cells with intact cell membrane. However, if the cells are damaged and cell membrane rupture occurs, PI agent can pass through the plasma membrane to bind with RNA and DNA in the cells, causing red fluorescence simultaneously (Khan Mohiuddin et al., 2010). Compared to PI, FDA is an organic compound that has no fluorescence and is nonpolar. It can pass in and out of the protoplast membrane freely. Because of the high esterase activity in vital living protoplast, FDA that enters into the cells will be broken down rapidly under the action of the esterase and form carboxyfluorescein (cF) which is difficult to penetrate cell membrane, and display green fluorescence. However, intracellular esterase outflow would occur, or protoplasts would lose its activity and possibly result in esterase inactivation, if the cell membrane is damaged. In this case, FDA would not excite fluorescence even if it entered into the cells. Wherefore, FDA can be used as a highly specific indicator for determining the cell activity and membrane integrity (Endo et al., 2000). We took advantage of PI and FDA dye for staining to detect necrotic cells and cell membrane integrity in each sample by FCM, with the tested bacterial cells being in logarithmic phase, stationary phase and decline phase, respectively. The results were shown in Fig. 3. Although S. maltophilia went through the stages from log phase to decline phase as incubation time passed, there were only a small amount of necrotic cells produced in different growth stages. In particular, only 8.2% of necrotic cells in the culture system were found when bacteria entered the decline phase. This was because there were no contaminants in the medium, the cell membrane kept intact even though the bacteria had already died, which made PI dye difficult to enter into the cells to link with DNA or RNA, consequently, no red fluorescence was displayed.

Also, the esterase inside cells did not leak and higher cell activity was maintained as the result of integral cell membrane. These results reflected the fact that the damage of bacterial cell membrane was due to the pollutants in the solution, rather than the metabolism and natural death of microorganism. 3.4. Effect of pollutants on cell membrane In order to analyze the alteration of cell membrane integrity and intracellular esterase activity after S. maltophilia decomposed/adsorbed BaP–Cu(II) combined contaminants, PI and FDA double staining method and FCM were used, and the results were shown in Fig. 4. It was seen that the cell membrane of S. maltophilia varied in different degree after interacting with BaP and Cu(II) for 2 d. Compared with BaP, more serious damage on cell membrane was caused by Cu(II) ions, with 28.3% of cells being detected displaying red fluorescence. The proportion of cells with red fluorescence in control and single BaP pollution system, by contrast, was 9.2% and 13.7%, separately. In addition, it was found that most of cells displayed green fluorescence, and the ratio of cells with high activity of carboxyfluorescein (cF) was over 90% in all systems, except one in which single Cu(II) contaminant was contained. This indicated that the intracellular esterase activity was not inhibited and still had higher activity to decompose/transform pollutants, even though the cell permeability had altered by contaminants, which even resulted in some membrane rupture and emerging of ‘‘holes’’ (see the first result of this study). Our previous studies have shown that the effect of different types of pollutants on cell membrane permeability of S. maltophilia was not the same (Chen et al., 2012). And it was also proved that the influence of heavy metal copper was more obvious, which was confirmed by our following investigation on cell microstructure in this study that both BaP–Cu(II) combined pollutants and single Cu(II) ions posed significantly higher impact on cell membrane than single BaP contaminant after 2 d. This made PI easier to get access to bacterial cells to combine with RNA in the presence of Cu(II) ions. On the other hand, our previous investigation also found that the mechanism of influence of Cu(II) ions on membrane permeability of S. maltophilia was by means of ion channels in cells. This was in agreement with our experimental result that the cell membrane integrity and intracellular esterase activity were not obviously affected. 3.5. Effect of pollutants on activity of ATP enzyme in bacterial cells ATP enzyme is a major biofilm protease that is ubiquitous in organism. It is very important, since the activity of ATP enzyme

cell proportion/ %

100

cell proportions/ %

120 100 80 60 40

60 40 20 0 control

20 0

80

Log

Sta growth phase

Necrotic cells

Dec Intact cells

Fig. 3. Cell activity and membrane integrity of S. maltophilia in different growth phase. The strain was cultivated in nutrient medium at 30 °C with shaking at 130 rmin1, and sampled at 12, 36 and 60 h, respectively.

BaP

Membrane damage

BaP-Cu samples

Cu

Esterase activity

Fig. 4. Cell membrane damage and esterase activity of S. maltophilia during BaP biodegradation and Cu(II) biosorption. BaP (1 mg L1), co-existed BaP (1 mg L1) and Cu(II) (10 mg L1), and Cu(II) (10 mg L1) in liquid MSM systems respectively were involved. Reactions were taken place at 30 °C with shaking at 130 r min1 for two days. The control system was without contaminant.

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is directly related to the transport and metabolism of various intracellular ions, thus affecting a variety of cellular functions. Microorganisms use ATP enzyme to produce ADP and inorganic phosphorus (Kim et al., 2013; Munshi et al., 2013; Tabachnikov and Shoham, 2013). In this experiment the amount of inorganic phosphorus inside cells and total protein of microorganism were measured individually, in order to characterize the ATP enzyme activity based on the amount of inorganic phosphate released for ATP decomposition per mg protein per min. The experimental results were presented in Fig.5. Generally speaking, the ATP activity in microbial cell will be influenced by the nutrients and pollutants in systems, and the bacterial metabolism will be inhibited by lack of energy substances. Compared with the performance in nutrition culture system (ck1), the activity of ATP enzyme in S. maltophilia in inorganic salt culture system (ck2) decreased significantly owing to an absence of carbon source needed for microbial growth in this medium. In the presence of contaminants, however, the bacterial cells would be induced to activate related enzymes. Thereupon, S. maltophilia utilized BaP as carbon source for metabolism, leading to the increase of ATP enzyme activity. As seen in Fig.5, the transport and metabolism of Na+/K+, Ca2+ and Mg2+ in S. maltophilia cells were affected during BaP degradation and Cu(II) adsorption, compared to those in nutrition and inorganic salt system. In particular, the Na+/K+-ATPase and Ca2+-ATPase activity were significantly improved when there was only BaP in the solution. On the contrary, the presence of Cu(II) in the system restrained the Ca2+-ATPase activity of microbial cells. This implied that Ca(II) ion metabolism in solution was interfered during Cu(II) removal by S. maltophilia (Ye et al., 2013), due to the fact that the transport pathway of divalent copper ions in microbial cells were similar to that of divalent calcium ions. Cu(II) ions in solution were adsorbed on the bacterial surface firstly, and then transferred into the cells by calcium channel on the membrane, thus preventing the metabolism of Ca(II) ions in cells. Mineral element magnesium is an essential biological nutrient. Mg(II) ions in bacterial cells can activate a variety of enzymes activity, most of which exist in the form of active Mg2+-ATPase. It was demonstrated that Mg(II) metabolism was influenced during BaP degradation and Cu(II) removal (Ye et al., 2013). This was because S. maltophilia had to start the functions of related intracellular proteins and degrading enzymes if it was designated to remove the contaminants, and Mg(II) ion was a key enzyme activator. Therefore, Mg2+-ATPase activity in bacterial cells increased owing to the induction by pollutants in the system.

ATPase activity (umolPi/mgprot/min)

160 140 120

3.6. Morphological analysis of S. maltophilia The morphology of microbial cells and their internal structures were prone to change or even damage when exposed to hazardous and toxic pollutants, causing the deficiency of their functions (Bruins et al., 2000; Keasling Jay, 2008). Therefore, the knowledge of microbial structures is of great importance to better understand the variation of microbial physiology. In this research, TEM was used to observe the internal morphology of S. maltophilia before and after dealing with BaP–Cu(II) combined pollutants for 2 d. The bacterial cells in system without contaminants were plump and had integral cell structure with uniform cytoplasm and obvious nucleus. And some cells even remained multinucleate. After contacting with pollutants for 2 d, especially in the presence of Cu(II) ions, the cell surface of S. maltophilia became rough, cytoplasm in core zone of cells appeared diffused, but some wounded filamentous genetic materials still remained, particularly, the structure of microbial cells still kept complete to protect key inclusions that would probably be crucial for BaP degradation and Cu(II) biotransformation from losing. That was why S. maltophilia could deal with BaP and Cu(II) pollutants over a long time. Furthermore, by comparing bacteria in single BaP, Cu(II) and BaP–Cu(II) combined pollutants systems, it could be found that the cell morphology had been affected more severely by Cu(II) ions in solution, with more seriously destroyed membrane and significantly less cell inclusion left. The experimental phenomena obtained here could also confirm the results presented in our previous section of this study that when S. maltophilia was exposed to contaminants, the permeability of cell membrane changed and some cells even ruptured, which made PI easily enter into cells to combine with RNA and display red fluorescence; nevertheless, the bacterial cells did not burst completely, which prevented the intracellular esterase from losing and kept the cells active. That was why FDA could still interact with esterase inside cells and emit green fluorescence after 2 d. The images of microstructure observation of S. maltophilia by TEM were included as Supplementary material. 4. Conclusions S. maltophilia was tolerant of co-existed BaP and Cu(II) that could be rapidly conveyed into cells, and BaP was stored in microbial cell first instead of being decomposed and used instantly. Although the presence of BaP and Cu(II) ion strengthened microbial membrane permeability and affected its morphology, the integrity of cell structure of S. maltophilia was still maintained, high intracellular esterase activity and genetic materials still remained through FCM analysis and microscopic observation. Moreover, the bacterial ions transport and metabolism were interfered, and the ATP enzymes activity was then enhanced, due to the influence of contaminants.

100 80

Acknowledgements

60 40 20 0 nutrient MSM(ck2) medium(ck1) Na/K-ATPase

BaP

BaP-Cu

Cu

samples Ca/Mg-ATPase

The authors would like to thank the National Natural Science Foundation of China (No. U0933002, 50978122, 41330639), Natural Science Foundation of Guangdong Province, China (S2013020012808), and the Fundamental Research Funds for the Central Universities (No. 2013ZM0126) for the financial support of this work.

Mg-ATPase

Fig. 5. Changes of activity of ATP enzyme in bacterial cells. BaP (1 mg L1), coexisted BaP (1 mg L1) and Cu(II) (10 mg L1), and Cu(II) (10 mg L1) in liquid MSM systems respectively were involved. Reactions were taken place at 30 °C with shaking at 130 r min1 for two days. The cells in blank control 1 (ck1) was grown at 30 °C for 2d in nutrient medium, while the blank control 2 system (ck2) was MSM without contaminants.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 02.020.

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