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Effects of inorganic salts on denitrifying granular sludge: The acute toxicity and working mechanisms
Bioresource Technology 204 (2016) 65–70
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effects of inorganic salts on denitrifying granular sludge: The acute toxicity and working mechanisms Ru Wang, Ping Zheng ⇑, A-qiang Ding, Meng Zhang, Abbas Ghulam, Cheng Yang, He-Ping Zhao Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, PR China
h i g h l i g h t s The IC50 of NaCl, Na2SO4 and Na3PO4 on DGS were 11.46, 21.72 and 7.46 g/L. The LC50 of NaCl, Na2SO4 and Na3PO4 on DGS were 77.35, 100.58 and 67.92 g/L. The toxicity of low and high salinity had different working mechanisms.
a r t i c l e
i n f o
Article history: Received 20 October 2015 Received in revised form 16 December 2015 Accepted 18 December 2015 Available online 23 December 2015 Keywords: Inorganic salts Denitrifying granular sludge Acute toxicity Activity inhibition Cell lethality
a b s t r a c t It is highly significant to investigate the toxicity of inorganic salts to denitrifying granular sludge (DGS) and its mechanism since the application of high-rate denitrification is seriously limited in the treatment of saline nitrogen-rich wastewaters. The batch experiments showed that the IC50 (half inhibition concentration) and LC50 (half lethal concentration) of NaCl, Na2SO4 and Na3PO4 on DGS were 11.46, 21.72, 7.46 g/L and 77.35, 100.58, 67.92 g/L respectively. Based on the analysis of specific denitrifying activity, the live cell percentage, the cell structure, and the DNA leakage, the toxicity of low salinity was ascribed to the inhibition of denitrifying activity and the toxicity of high salinity was ascribed to both the inhibition of denitrifying activity and the lethality of denitrifying cell. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Denitrification is widely applied in the biological nitrogen removal from wastewaters, but limited in the treatment of saline nitrogen-rich wastewaters (Yu et al., 2012; Wen et al., 1999). The saline nitrogen-rich wastewaters contain high nitrogen pollutants and inorganic salts. Many inorganic salts exist in natural ecoenvironment at relatively low concentrations, while the industrial wastewaters have only several inorganic salts with much higher concentrations (Glass and Silverstein, 1999; Wan et al., 2014; Lefebvre and Moletta, 2006). Especially nowadays, some industrial factories have promoted their outputs to meet the market demands which results in vast high-salinity nitrogen-rich wastewaters. For example, it has been reported that the chlorate concentration in P-aminoazobenzenic salt production wastewater from a printing and dyeing plant was up to 100 g/L; the sulfate concentration in metal processing wastewater from a steel mill was up to 120 g/L; the phosphate concentration in imidazole aldehyde ⇑ Corresponding author. Tel./fax: +86 0571 88982819. E-mail address: [email protected] (P. Zheng). http://dx.doi.org/10.1016/j.biortech.2015.12.062 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
hydrolyzation wastewater from a pharmaceutical factory was up to 95 g/L (Hamoda and Al-Attar, 1995; Ramos et al., 2007; Chowdhury et al., 2010). Every microbial species has its optimum growth salinity, and the microorganism would lose its activity beyond the tolerant limit (Shen et al., 2015). Halophiles enjoy the higher salinity than nonhalophiles, and the separatrix of salinity (NaCl) for halophiles from non-halophiles was 17 g/L (Ollivier et al., 1994). Though marine microorganisms were considered as best inocula to start up the salinity-tolerant denitrifying reactor since halophiles are widely spread in seawaters (Babatsouli et al., 2015; Duan et al., 2015), it was not realistic to get the marine microorganisms in a large amount, thus highly active denitrifying sludge was often acclimatized to treat the saline nitrogen-rich wastewaters. The denitrifying sludges could display good performances at low salinities (620 g/L) after acclimation, but not at high salinities (Miao et al., 2015; Yoshie et al., 2006). Researchers found that the denitrifying community changed greatly, sometimes even lost function, after exposure to high salinity. Yu et al. reported that Marinobacter dominated in denitrifying reactor with influent NaCl concentration of 110 g/L (Miao et al., 2015). Yoshie et al. found that Halomonas
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dominated in denitrifying reactor with NaCl concentration of 40–100 g/L (Yoshie et al., 2006). So far, little information is available about the toxicity of inorganic salts to denitrifying sludge and its mechanism. In the practical application of high-rate denitrifying reactor, the large fluctuation of salinity might cause salinity shock, and lead to the collapse of denitrifying reactor (Duan et al., 2015; Liu et al., 2009). Therefore, it is important to investigate the toxicity of inorganic salts to denitrifying granular sludge (DGS) and understand the working mechanism. In this work, NaCl, Na2SO4 and Na3PO4 were chosen as representative salts of industrial wastewaters; their inhibition and lethality to DGS were evaluated by determining the specific denitrifying activity and the live cell percentage; and their mechanisms of toxicity were researched by observing cell structure and DNA leakage. All results obtained in this paper were used to guide the development of salinity-tolerant high-rate denitrifying reactor. 2. Methods
where r s was the specific denitrifying activity (g N/(g VSS h)), Dc was the change of nitrate concentration (g N/L), m was the biomass concentration (g VSS/L) and t was the reaction time (h). 2.4. Determination of live cell percentage After exposure to inorganic salts for 5 h, DGS was dispersed by ultrasonication to obtain cell suspension. Live/DeadÒ BaclightTM Bacterial Viability Kit (Molecular Probes, USA) was used to stain the nucleic acid so as to distinguish the live cells from the dead cells. The fluorescence microscope (Leica, Germany) was used to observe and photograph the cells. Image-Pro Plus 6.0 was used to count the number of live and dead cells (Wu and Xi, 2009). 2.5. Determination of DNA leakage After exposure to inorganic salts for 5 h, the supernatant was sampled and filtrated through 0.22 lm membrane. Then, the absorbance of filtrate was determined at 260 nm which was the characteristic absorption peak of DNA.
2.1. Denitrifying granular sludge The denitrifying granular sludge (DGS) was taken from a labscale high-rate denitrifying reactor which was fed with sodium nitrate as electron accepter and methanol as electron donor. The nitrate loading rate of the reactor was 35.14 ± 0.38 kg N/(m3 d) with hydraulic retention time of 7 h. The value of C/N in influent was 1:3.33, and a fixed recycling ratio of 2.0 was set to dilute the influent. The total solids (TS) and volatile suspended solid (VSS) of DGS were 136.68 g/L and 52.89 g/L, respectively (Li et al., 2013). 2.2. Synthetic wastewater Based on the basal medium, series of salt concentrations were set to investigate the impacts of NaCl, Na2SO4 and Na3PO4 on DGS. According to the salt concentration in industrial wastewaters, the upper limit concentrations of NaCl, Na2SO4 and Na3PO4 were determined as 100, 120, 95 g/L respectively. Then, different concentrations of NaCl were set as 6, 14, 20, 40, 60, 80, 100 g/L. Different concentrations of Na2SO4 were set as 7, 14, 24, 48, 72, 96, 120 g/L. Different concentrations of Na3PO4 were set as 6, 13, 19, 38, 57, 76, 95 g/L. The basal medium contained: NaNO3 0.61 g/L, CH3COONa 0.64 g/L, KH2PO3 0.05 g/L, CaCl2 0.04 g/L, MgSO47H2O 0.01 g/L, and 1 ml/L of trace element solution. The components of trace element solution were: EDTA 5 g/L, MnCl24H2O 5 g/L, FeSO47H2O 3 g/L, CoCl26H2O 0.05 g/L, NiCl2 6H2O 0.04 g/L, H3BO3 0.02 g/L, (NH4)6Mo7O24H2O 0.02 g/L, CuSO4 5H2O 0.01 g/L and ZnSO4 0.003 g/L. 2.3. Determination of specific denitrifying activity To test the inhibition of inorganic salts to the specific denitrifying activity, batch experiments were conducted in serum bottles with butyl rubber stoppers and aluminum caps (Wang et al., 2014). After washing three times by 0.9% NaCl solution, 5 g DGS (wet sludge) was put into 50 mL mineral medium with different salt concentrations. The initial concentrations of COD and NO 3 -N were set at 5000 mg/l and 100 mg/l, respectively. Experimental groups were added with different concentrations of NaCl or Na2SO4 or Na3PO4 respectively, while bottles without adding any inorganic salt were set as control. All tests were cultivated on a shaking table (120 rpm) at 30 °C for 5 h, conducted in duplicate. The specific denitrifying activity was calculated by Eq. (1).
r s ¼ Dc=ðm tÞ
ð1Þ
2.6. Observation of cell structure DGS sample was fixed with 2.5% buffering glutaraldehyde for 12 h at 4 °C, then post-fixed in 1% buffering osmium tetroxide for 1 h, stained with 1% uranyl acetate and dehydrated in a series of ethanol. After that, DGS sample was embedded by embedding medium for a whole night. Ultra-thin sections were prepared and stained with 1% uranyl acetate and sodium citrate. Microscopy was carried out with a Hitachi H-7650 (Tokyo, Japan) microscope (transmission electron microscope, TEM). 2.7. Analytical methods All samples were measured immediately after sampling. The concentrations of nitrate and nitrite were determined according to the standard methods (APHA, 2005). The pH values were determined by a S20 K pH meter (Mettler Toledo, Switzerland). 3. Results and discussion 3.1. Determination of half inhibitory concentration Batch experiments were conducted to test the effects of NaCl, Na2SO4 and Na3PO4 on the specific denitrifying activity of DGS. As shown in Fig. 1A–C, the trends of specific denitrifying activity versus salts concentrations could be easily divided into two sections. In the first section, the specific denitrifying activity decreased linearly; while in the second section, it leveled off along with the increase of salt concentration. The cut-off point of two sections was 20 g/L NaCl (24 g/L Na2SO4, 19 g/L Na3PO4). Hereafter, the salinity with 0–20 g/L NaCl (0–24 g/L Na2SO4, 0–19 g/L Na3PO4) was defined as the low salinity, while the salinity with 20–100 g/L NaCl (24–120 g/L Na2SO4, 19–95 g/L Na3PO4) was defined as the high salinity. At the low salinity (620 g/L for NaCl, 24 g/L for Na2SO4, 19 g/L for Na3PO4), the decrease of specific denitrifying activity was 20.47 g N/(g VSS h) for NaCl, 16.44 g N/(g VSS h) for Na2SO4, and 21.69 g N/(g VSS h) for Na3PO4, relatively decreased 73.73% for NaCl, 59.21% for Na2SO4, and 78.09% for Na3PO4 comparing with the control. At the high salinity (20–100 g/L for NaCl, 24–120 g/L for Na2SO4, 19–95 g/L for Na3PO4), the decrease of specific denitrifying activity was 27.69 g N/(g VSS h) for NaCl, 26.50 g N/(g VSS h) for Na2SO4, and 27.56 g N/(g VSS h) for Na3PO4, relatively decreased 99.72% for NaCl, 95.42% for Na2SO4, and 99.27% for
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Fig. 1. The curve of specific denitrifying activity versus concentration of different salts.
Na3PO4 comparing with the control. Both in the groups of low salinity and high salinity, no nitrite were detected. Eq. (2) (derived in Supplementary material) was used to fit the data of Fig. 1D (Henze et al., 2010).
rS ¼
kK I K I þ SI
ð2Þ
where r S was the specific denitrifying activity [g N/(g VSS h)], K I was the IC50 (g/L), k was the reaction constant, and SI was the concentration of inorganic salt (g/L). Based on the regression equation, the half inhibition concentrations (IC50) for NaCl, Na2SO4 and Na3PO4 on the specific denitrifying activity of DGS were obtained as 11.46, 21.72, 7.46 g/L, which were close to the reported numbers (Mariangel et al., 2008; Panswad and Anan, 1999). According to the IC50 values, domestication of denitrifying microorganisms might be useful for the low-salinity wastewater treatment. However for the high-salinity wastewater, the activity of denitrifying microorganisms was entirely inhibited at the very start. Dilution of influent and reformation of denitrifying microorganisms might be the solutions.
field of fluorescence microscope, and the live cell percentages decreased to 37.11% for NaCl, 39.63% for Na2SO4, and 34.39% for Na3PO4. In a word, more cells of DGS died along with the increment of salinity. Comparatively, the live cell percentage was 99.60% in the control. Taking the value of control as 100%, the relative live cell percentages of experimental groups were calculated and shown in Fig. 2A–C. At the low salinity (620 g/L for NaCl, 24 g/L for Na2 SO4, 19 g/L for Na3PO4), the relative live cell percentages were stable as in the control (decreased 0.20% for NaCl, 0.61% for Na2SO4 and 14.13% for Na3PO4 only). On the contrary, at the high salinity (20–100 g/L for NaCl, 24–120 g/L for Na2SO4, 19–95 g/L for Na3 PO4), the relative live cell percentages decreased linearly along with the increase of salts concentrations (62.50% for NaCl, 59.97% for Na2SO4 and 65.22% for Na3PO4). The half lethal concentrations (LC50) of NaCl, Na2SO4 and Na3PO4 on the cells in DGS were 77.35, 100.58 and 67.92 g/L respectively (shown in Fig. 2D). According to the LC50 values, most of cells were alive even exposure to the high salinity. That meant the recovery of denitrifying reactor is possible after the salinity shock.
3.2. Determination of half lethal concentration
3.3. Damage of cell structure
Fig. S1 showed the fluorescence microscopic images of cells in DGS being stained by the Live/DeadÒ BaclightTM Bacterial Viability Kit. After exposure to the low salinity (20 g/L NaCl, 24 g/L Na2SO4, 19 g/L Na3PO4) for 5 h, the cells took on green in the visual field of fluorescence microscope, and the live cell percentages were 99.40% for NaCl, 98.99% for Na2SO4, and 85.48% for Na3PO4. Differently, after exposure to the high salinity (100 g/L NaCl, 120 g/L Na2SO4, 95 g/L Na3PO4) for 5 h, however, the cells turned red in the visual
Fig. S2 showed the TEM photographs of cell structures. As displayed in Fig. S2-A1–C1, no significant change of cell structure was observed after exposure to the low salinity (620 g/L for NaCl, 24 g/L for Na2SO4, 19 g/L for Na3PO4) for 5 h. On the contrary, as shown in Fig. S2-A2–C2, a large change of cell structure was observed after exposure to the high salinity (20–100 g/L for NaCl, 24–120 g/L for Na2SO4, 19–95 g/L for Na3PO4) for 5 h, the protoplast shrunk, the periplasmic space became larger, the cell
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Fig. 2. The relative live cell percentage versus concentration of different salt.
membrane broke and the cell components including DNA leaked out. However, the spherical cell held an intact cell membrane, with nucleoid in the center and cytoplasm dispersed evenly in the control (Data not shown). 3.4. DNA leakage out of cell membrane DNA located inside the cell and can be detected only when the cell membrane’s damaged. So, the DNA leakage could characterize the cell membrane damage from inorganic salts. Taking the DNA leakage in the control as 100%, the relative DNA leakages in the experimental groups were calculated and shown in Fig. 3A–C. At the low salinity (620 g/L for NaCl, 24 g/L for Na2SO4, 19 g/L for Na3 PO4), the relative DNA leakages were almost stable as in the control (123.33% for NaCl, 115.83% for Na2SO4, and 153.33% for Na3PO4). At the high salinity (20–100 g/L for NaCl, 24–120 g/L for Na2SO4, 19–95 g/L for Na3PO4), on the contrary, the relative DNA leakage increased linearly along with the increase of salts concentrations (571.67% for NaCl, 792.50% for Na2SO4 and 686.67% for Na3PO4) (shown in Fig. 3D). The DNA leakages resulted from the inorganic salts confirmed the damage of cell structure shown in Section 3.3. 3.5. Analysis of toxicity mechanism for inorganic salts According to the results in this work, the toxicity of inorganic salts to the cells in DGS was acute, and the toxicity mechanism was obviously different for low salinity and high salinity. The denitrifying activity and the live cell number are two key factors that determine the denitrifying capacity of DGS. The denitrifying activity of DGS can be characterized by the specific denitrifying activity, while the live cell number can be characterized by the live cell percentage. Both the denitrifying activity and the live cell number depend on the integral cell structure, thus the damage of cell
structure and the DNA leakage can help to explain the inhibition to denitrifying activity and the lethality to denitrifying cells. At the low salinity, the specific denitrifying activity of DGS decreased linearly, while the live cell percentage was almost constant. The cell structure in DGS was intact (normal), and the DNA leakage was negligible. Based on the analysis, the effect of low salinity on the denitrifying capacity of DGS could be ascribed to the inhibition of inorganic salts to denitrifying activity of DGS, namely the inhibition on the functional enzymes. Thauera and Hyphomicrobium, gram negative denitrifiers, were found to dominate in DGS (Fig. S3 and Table S1) (Martineau et al., 2013; Scholten et al., 1999). In these denitrifiers, the nitrite reductase and nitrous reductase are located in periplasm (Fig. 4A). The active center of nitrate reductase integrated in the cell membrane facing to periplasmic space (Kraft et al., 2011; Sparacino-Watkins et al., 2014). When the cells in DGS were exposed to saline environment, ions entered into the periplasmic space through pore proteins inlayed in the outer cell membrane. The interflow between the extracellular environment and the intracellular environment made the salt concentration in periplasm space to be the same as that in outer environment (Cabello et al., 2004). As soon as subjected to the inorganic salts, the denitrifying enzymes began to lose their activities because of salting-out (Fig. 4B). At the high salinity, the specific denitrifying activity of DGS decreased largely, and the live cell percentage decreased simultaneously. The cells in DGS were broken with damaged cell membrane, and the DNA leakage became larger with the increase of salt concentration. Based on the analysis, the effect of high salinity on the denitrifying capacity of DGS could be ascribed to both the inhibition to denitrifying activity and the lethality to denitrifying cells. At the high salinity, the ions could accumulate a large electrostatic force that was strong enough to break the cell structure including the cell wall and cell membrane, which led to the leakage
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Fig. 3. The relative DNA leakage versus concentration of different salt.
4. Conclusion The toxicity of NaCl, Na2SO4 and Na3PO4 to the microbial cells in DGS was acute, the IC50 of NaCl, Na2SO4 and Na3PO4 on the specific denitrifying activity of DGS was 11.46, 21.72, 7.46 g/L, and the LC50 of NaCl, Na2SO4 and Na3PO4 on the cells in DGS was 77.35, 100.58 and 67.92 g/L. Effects of low salinity on the denitrifying capacity of DGS were ascribed to the inhibition of inorganic salts to denitrifying activity, and effects of high salinity on the denitrifying capacity of DGS were ascribed to both the inhibition to denitrifying activity and the lethality to denitrifying cells. Acknowledgements This work was financially supported by National Natural Science Foundation of China (51278457), National Key Technology R&D Program of China (2013BAD21B04) and Key Science and Technology Innovation Team Grant of Zhejiang (2013TD12). Appendix A. Supplementary data Fig. 4. Toxicity mechanism for inorganic salts to DGS.
of cell components including DNA (Mendis et al., 2000). In addition, the ions at the high salinity could denature the cell components such as enzymes and nucleoids, and destroy the cell activity by salting-out effect (Fig. 4B) (Zhao, 2005). As shown in Fig. S4, the activity of alkaline phosphatase (one of the endoenzymes) was inhibited linearly with the increase of inorganic salt concentration.
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effects of inorganic salts on denitrifying granular sludge: The acute toxicity and working mechanisms Ru Wang, Ping Zheng ⇑, A-qiang Ding, Meng Zhang, Abbas Ghulam, Cheng Yang, He-Ping Zhao Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, PR China
h i g h l i g h t s The IC50 of NaCl, Na2SO4 and Na3PO4 on DGS were 11.46, 21.72 and 7.46 g/L. The LC50 of NaCl, Na2SO4 and Na3PO4 on DGS were 77.35, 100.58 and 67.92 g/L. The toxicity of low and high salinity had different working mechanisms.
a r t i c l e
i n f o
Article history: Received 20 October 2015 Received in revised form 16 December 2015 Accepted 18 December 2015 Available online 23 December 2015 Keywords: Inorganic salts Denitrifying granular sludge Acute toxicity Activity inhibition Cell lethality
a b s t r a c t It is highly significant to investigate the toxicity of inorganic salts to denitrifying granular sludge (DGS) and its mechanism since the application of high-rate denitrification is seriously limited in the treatment of saline nitrogen-rich wastewaters. The batch experiments showed that the IC50 (half inhibition concentration) and LC50 (half lethal concentration) of NaCl, Na2SO4 and Na3PO4 on DGS were 11.46, 21.72, 7.46 g/L and 77.35, 100.58, 67.92 g/L respectively. Based on the analysis of specific denitrifying activity, the live cell percentage, the cell structure, and the DNA leakage, the toxicity of low salinity was ascribed to the inhibition of denitrifying activity and the toxicity of high salinity was ascribed to both the inhibition of denitrifying activity and the lethality of denitrifying cell. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Denitrification is widely applied in the biological nitrogen removal from wastewaters, but limited in the treatment of saline nitrogen-rich wastewaters (Yu et al., 2012; Wen et al., 1999). The saline nitrogen-rich wastewaters contain high nitrogen pollutants and inorganic salts. Many inorganic salts exist in natural ecoenvironment at relatively low concentrations, while the industrial wastewaters have only several inorganic salts with much higher concentrations (Glass and Silverstein, 1999; Wan et al., 2014; Lefebvre and Moletta, 2006). Especially nowadays, some industrial factories have promoted their outputs to meet the market demands which results in vast high-salinity nitrogen-rich wastewaters. For example, it has been reported that the chlorate concentration in P-aminoazobenzenic salt production wastewater from a printing and dyeing plant was up to 100 g/L; the sulfate concentration in metal processing wastewater from a steel mill was up to 120 g/L; the phosphate concentration in imidazole aldehyde ⇑ Corresponding author. Tel./fax: +86 0571 88982819. E-mail address: [email protected] (P. Zheng). http://dx.doi.org/10.1016/j.biortech.2015.12.062 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
hydrolyzation wastewater from a pharmaceutical factory was up to 95 g/L (Hamoda and Al-Attar, 1995; Ramos et al., 2007; Chowdhury et al., 2010). Every microbial species has its optimum growth salinity, and the microorganism would lose its activity beyond the tolerant limit (Shen et al., 2015). Halophiles enjoy the higher salinity than nonhalophiles, and the separatrix of salinity (NaCl) for halophiles from non-halophiles was 17 g/L (Ollivier et al., 1994). Though marine microorganisms were considered as best inocula to start up the salinity-tolerant denitrifying reactor since halophiles are widely spread in seawaters (Babatsouli et al., 2015; Duan et al., 2015), it was not realistic to get the marine microorganisms in a large amount, thus highly active denitrifying sludge was often acclimatized to treat the saline nitrogen-rich wastewaters. The denitrifying sludges could display good performances at low salinities (620 g/L) after acclimation, but not at high salinities (Miao et al., 2015; Yoshie et al., 2006). Researchers found that the denitrifying community changed greatly, sometimes even lost function, after exposure to high salinity. Yu et al. reported that Marinobacter dominated in denitrifying reactor with influent NaCl concentration of 110 g/L (Miao et al., 2015). Yoshie et al. found that Halomonas
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dominated in denitrifying reactor with NaCl concentration of 40–100 g/L (Yoshie et al., 2006). So far, little information is available about the toxicity of inorganic salts to denitrifying sludge and its mechanism. In the practical application of high-rate denitrifying reactor, the large fluctuation of salinity might cause salinity shock, and lead to the collapse of denitrifying reactor (Duan et al., 2015; Liu et al., 2009). Therefore, it is important to investigate the toxicity of inorganic salts to denitrifying granular sludge (DGS) and understand the working mechanism. In this work, NaCl, Na2SO4 and Na3PO4 were chosen as representative salts of industrial wastewaters; their inhibition and lethality to DGS were evaluated by determining the specific denitrifying activity and the live cell percentage; and their mechanisms of toxicity were researched by observing cell structure and DNA leakage. All results obtained in this paper were used to guide the development of salinity-tolerant high-rate denitrifying reactor. 2. Methods
where r s was the specific denitrifying activity (g N/(g VSS h)), Dc was the change of nitrate concentration (g N/L), m was the biomass concentration (g VSS/L) and t was the reaction time (h). 2.4. Determination of live cell percentage After exposure to inorganic salts for 5 h, DGS was dispersed by ultrasonication to obtain cell suspension. Live/DeadÒ BaclightTM Bacterial Viability Kit (Molecular Probes, USA) was used to stain the nucleic acid so as to distinguish the live cells from the dead cells. The fluorescence microscope (Leica, Germany) was used to observe and photograph the cells. Image-Pro Plus 6.0 was used to count the number of live and dead cells (Wu and Xi, 2009). 2.5. Determination of DNA leakage After exposure to inorganic salts for 5 h, the supernatant was sampled and filtrated through 0.22 lm membrane. Then, the absorbance of filtrate was determined at 260 nm which was the characteristic absorption peak of DNA.
2.1. Denitrifying granular sludge The denitrifying granular sludge (DGS) was taken from a labscale high-rate denitrifying reactor which was fed with sodium nitrate as electron accepter and methanol as electron donor. The nitrate loading rate of the reactor was 35.14 ± 0.38 kg N/(m3 d) with hydraulic retention time of 7 h. The value of C/N in influent was 1:3.33, and a fixed recycling ratio of 2.0 was set to dilute the influent. The total solids (TS) and volatile suspended solid (VSS) of DGS were 136.68 g/L and 52.89 g/L, respectively (Li et al., 2013). 2.2. Synthetic wastewater Based on the basal medium, series of salt concentrations were set to investigate the impacts of NaCl, Na2SO4 and Na3PO4 on DGS. According to the salt concentration in industrial wastewaters, the upper limit concentrations of NaCl, Na2SO4 and Na3PO4 were determined as 100, 120, 95 g/L respectively. Then, different concentrations of NaCl were set as 6, 14, 20, 40, 60, 80, 100 g/L. Different concentrations of Na2SO4 were set as 7, 14, 24, 48, 72, 96, 120 g/L. Different concentrations of Na3PO4 were set as 6, 13, 19, 38, 57, 76, 95 g/L. The basal medium contained: NaNO3 0.61 g/L, CH3COONa 0.64 g/L, KH2PO3 0.05 g/L, CaCl2 0.04 g/L, MgSO47H2O 0.01 g/L, and 1 ml/L of trace element solution. The components of trace element solution were: EDTA 5 g/L, MnCl24H2O 5 g/L, FeSO47H2O 3 g/L, CoCl26H2O 0.05 g/L, NiCl2 6H2O 0.04 g/L, H3BO3 0.02 g/L, (NH4)6Mo7O24H2O 0.02 g/L, CuSO4 5H2O 0.01 g/L and ZnSO4 0.003 g/L. 2.3. Determination of specific denitrifying activity To test the inhibition of inorganic salts to the specific denitrifying activity, batch experiments were conducted in serum bottles with butyl rubber stoppers and aluminum caps (Wang et al., 2014). After washing three times by 0.9% NaCl solution, 5 g DGS (wet sludge) was put into 50 mL mineral medium with different salt concentrations. The initial concentrations of COD and NO 3 -N were set at 5000 mg/l and 100 mg/l, respectively. Experimental groups were added with different concentrations of NaCl or Na2SO4 or Na3PO4 respectively, while bottles without adding any inorganic salt were set as control. All tests were cultivated on a shaking table (120 rpm) at 30 °C for 5 h, conducted in duplicate. The specific denitrifying activity was calculated by Eq. (1).
r s ¼ Dc=ðm tÞ
ð1Þ
2.6. Observation of cell structure DGS sample was fixed with 2.5% buffering glutaraldehyde for 12 h at 4 °C, then post-fixed in 1% buffering osmium tetroxide for 1 h, stained with 1% uranyl acetate and dehydrated in a series of ethanol. After that, DGS sample was embedded by embedding medium for a whole night. Ultra-thin sections were prepared and stained with 1% uranyl acetate and sodium citrate. Microscopy was carried out with a Hitachi H-7650 (Tokyo, Japan) microscope (transmission electron microscope, TEM). 2.7. Analytical methods All samples were measured immediately after sampling. The concentrations of nitrate and nitrite were determined according to the standard methods (APHA, 2005). The pH values were determined by a S20 K pH meter (Mettler Toledo, Switzerland). 3. Results and discussion 3.1. Determination of half inhibitory concentration Batch experiments were conducted to test the effects of NaCl, Na2SO4 and Na3PO4 on the specific denitrifying activity of DGS. As shown in Fig. 1A–C, the trends of specific denitrifying activity versus salts concentrations could be easily divided into two sections. In the first section, the specific denitrifying activity decreased linearly; while in the second section, it leveled off along with the increase of salt concentration. The cut-off point of two sections was 20 g/L NaCl (24 g/L Na2SO4, 19 g/L Na3PO4). Hereafter, the salinity with 0–20 g/L NaCl (0–24 g/L Na2SO4, 0–19 g/L Na3PO4) was defined as the low salinity, while the salinity with 20–100 g/L NaCl (24–120 g/L Na2SO4, 19–95 g/L Na3PO4) was defined as the high salinity. At the low salinity (620 g/L for NaCl, 24 g/L for Na2SO4, 19 g/L for Na3PO4), the decrease of specific denitrifying activity was 20.47 g N/(g VSS h) for NaCl, 16.44 g N/(g VSS h) for Na2SO4, and 21.69 g N/(g VSS h) for Na3PO4, relatively decreased 73.73% for NaCl, 59.21% for Na2SO4, and 78.09% for Na3PO4 comparing with the control. At the high salinity (20–100 g/L for NaCl, 24–120 g/L for Na2SO4, 19–95 g/L for Na3PO4), the decrease of specific denitrifying activity was 27.69 g N/(g VSS h) for NaCl, 26.50 g N/(g VSS h) for Na2SO4, and 27.56 g N/(g VSS h) for Na3PO4, relatively decreased 99.72% for NaCl, 95.42% for Na2SO4, and 99.27% for
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Fig. 1. The curve of specific denitrifying activity versus concentration of different salts.
Na3PO4 comparing with the control. Both in the groups of low salinity and high salinity, no nitrite were detected. Eq. (2) (derived in Supplementary material) was used to fit the data of Fig. 1D (Henze et al., 2010).
rS ¼
kK I K I þ SI
ð2Þ
where r S was the specific denitrifying activity [g N/(g VSS h)], K I was the IC50 (g/L), k was the reaction constant, and SI was the concentration of inorganic salt (g/L). Based on the regression equation, the half inhibition concentrations (IC50) for NaCl, Na2SO4 and Na3PO4 on the specific denitrifying activity of DGS were obtained as 11.46, 21.72, 7.46 g/L, which were close to the reported numbers (Mariangel et al., 2008; Panswad and Anan, 1999). According to the IC50 values, domestication of denitrifying microorganisms might be useful for the low-salinity wastewater treatment. However for the high-salinity wastewater, the activity of denitrifying microorganisms was entirely inhibited at the very start. Dilution of influent and reformation of denitrifying microorganisms might be the solutions.
field of fluorescence microscope, and the live cell percentages decreased to 37.11% for NaCl, 39.63% for Na2SO4, and 34.39% for Na3PO4. In a word, more cells of DGS died along with the increment of salinity. Comparatively, the live cell percentage was 99.60% in the control. Taking the value of control as 100%, the relative live cell percentages of experimental groups were calculated and shown in Fig. 2A–C. At the low salinity (620 g/L for NaCl, 24 g/L for Na2 SO4, 19 g/L for Na3PO4), the relative live cell percentages were stable as in the control (decreased 0.20% for NaCl, 0.61% for Na2SO4 and 14.13% for Na3PO4 only). On the contrary, at the high salinity (20–100 g/L for NaCl, 24–120 g/L for Na2SO4, 19–95 g/L for Na3 PO4), the relative live cell percentages decreased linearly along with the increase of salts concentrations (62.50% for NaCl, 59.97% for Na2SO4 and 65.22% for Na3PO4). The half lethal concentrations (LC50) of NaCl, Na2SO4 and Na3PO4 on the cells in DGS were 77.35, 100.58 and 67.92 g/L respectively (shown in Fig. 2D). According to the LC50 values, most of cells were alive even exposure to the high salinity. That meant the recovery of denitrifying reactor is possible after the salinity shock.
3.2. Determination of half lethal concentration
3.3. Damage of cell structure
Fig. S1 showed the fluorescence microscopic images of cells in DGS being stained by the Live/DeadÒ BaclightTM Bacterial Viability Kit. After exposure to the low salinity (20 g/L NaCl, 24 g/L Na2SO4, 19 g/L Na3PO4) for 5 h, the cells took on green in the visual field of fluorescence microscope, and the live cell percentages were 99.40% for NaCl, 98.99% for Na2SO4, and 85.48% for Na3PO4. Differently, after exposure to the high salinity (100 g/L NaCl, 120 g/L Na2SO4, 95 g/L Na3PO4) for 5 h, however, the cells turned red in the visual
Fig. S2 showed the TEM photographs of cell structures. As displayed in Fig. S2-A1–C1, no significant change of cell structure was observed after exposure to the low salinity (620 g/L for NaCl, 24 g/L for Na2SO4, 19 g/L for Na3PO4) for 5 h. On the contrary, as shown in Fig. S2-A2–C2, a large change of cell structure was observed after exposure to the high salinity (20–100 g/L for NaCl, 24–120 g/L for Na2SO4, 19–95 g/L for Na3PO4) for 5 h, the protoplast shrunk, the periplasmic space became larger, the cell
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Fig. 2. The relative live cell percentage versus concentration of different salt.
membrane broke and the cell components including DNA leaked out. However, the spherical cell held an intact cell membrane, with nucleoid in the center and cytoplasm dispersed evenly in the control (Data not shown). 3.4. DNA leakage out of cell membrane DNA located inside the cell and can be detected only when the cell membrane’s damaged. So, the DNA leakage could characterize the cell membrane damage from inorganic salts. Taking the DNA leakage in the control as 100%, the relative DNA leakages in the experimental groups were calculated and shown in Fig. 3A–C. At the low salinity (620 g/L for NaCl, 24 g/L for Na2SO4, 19 g/L for Na3 PO4), the relative DNA leakages were almost stable as in the control (123.33% for NaCl, 115.83% for Na2SO4, and 153.33% for Na3PO4). At the high salinity (20–100 g/L for NaCl, 24–120 g/L for Na2SO4, 19–95 g/L for Na3PO4), on the contrary, the relative DNA leakage increased linearly along with the increase of salts concentrations (571.67% for NaCl, 792.50% for Na2SO4 and 686.67% for Na3PO4) (shown in Fig. 3D). The DNA leakages resulted from the inorganic salts confirmed the damage of cell structure shown in Section 3.3. 3.5. Analysis of toxicity mechanism for inorganic salts According to the results in this work, the toxicity of inorganic salts to the cells in DGS was acute, and the toxicity mechanism was obviously different for low salinity and high salinity. The denitrifying activity and the live cell number are two key factors that determine the denitrifying capacity of DGS. The denitrifying activity of DGS can be characterized by the specific denitrifying activity, while the live cell number can be characterized by the live cell percentage. Both the denitrifying activity and the live cell number depend on the integral cell structure, thus the damage of cell
structure and the DNA leakage can help to explain the inhibition to denitrifying activity and the lethality to denitrifying cells. At the low salinity, the specific denitrifying activity of DGS decreased linearly, while the live cell percentage was almost constant. The cell structure in DGS was intact (normal), and the DNA leakage was negligible. Based on the analysis, the effect of low salinity on the denitrifying capacity of DGS could be ascribed to the inhibition of inorganic salts to denitrifying activity of DGS, namely the inhibition on the functional enzymes. Thauera and Hyphomicrobium, gram negative denitrifiers, were found to dominate in DGS (Fig. S3 and Table S1) (Martineau et al., 2013; Scholten et al., 1999). In these denitrifiers, the nitrite reductase and nitrous reductase are located in periplasm (Fig. 4A). The active center of nitrate reductase integrated in the cell membrane facing to periplasmic space (Kraft et al., 2011; Sparacino-Watkins et al., 2014). When the cells in DGS were exposed to saline environment, ions entered into the periplasmic space through pore proteins inlayed in the outer cell membrane. The interflow between the extracellular environment and the intracellular environment made the salt concentration in periplasm space to be the same as that in outer environment (Cabello et al., 2004). As soon as subjected to the inorganic salts, the denitrifying enzymes began to lose their activities because of salting-out (Fig. 4B). At the high salinity, the specific denitrifying activity of DGS decreased largely, and the live cell percentage decreased simultaneously. The cells in DGS were broken with damaged cell membrane, and the DNA leakage became larger with the increase of salt concentration. Based on the analysis, the effect of high salinity on the denitrifying capacity of DGS could be ascribed to both the inhibition to denitrifying activity and the lethality to denitrifying cells. At the high salinity, the ions could accumulate a large electrostatic force that was strong enough to break the cell structure including the cell wall and cell membrane, which led to the leakage
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Fig. 3. The relative DNA leakage versus concentration of different salt.
4. Conclusion The toxicity of NaCl, Na2SO4 and Na3PO4 to the microbial cells in DGS was acute, the IC50 of NaCl, Na2SO4 and Na3PO4 on the specific denitrifying activity of DGS was 11.46, 21.72, 7.46 g/L, and the LC50 of NaCl, Na2SO4 and Na3PO4 on the cells in DGS was 77.35, 100.58 and 67.92 g/L. Effects of low salinity on the denitrifying capacity of DGS were ascribed to the inhibition of inorganic salts to denitrifying activity, and effects of high salinity on the denitrifying capacity of DGS were ascribed to both the inhibition to denitrifying activity and the lethality to denitrifying cells. Acknowledgements This work was financially supported by National Natural Science Foundation of China (51278457), National Key Technology R&D Program of China (2013BAD21B04) and Key Science and Technology Innovation Team Grant of Zhejiang (2013TD12). Appendix A. Supplementary data Fig. 4. Toxicity mechanism for inorganic salts to DGS.
of cell components including DNA (Mendis et al., 2000). In addition, the ions at the high salinity could denature the cell components such as enzymes and nucleoids, and destroy the cell activity by salting-out effect (Fig. 4B) (Zhao, 2005). As shown in Fig. S4, the activity of alkaline phosphatase (one of the endoenzymes) was inhibited linearly with the increase of inorganic salt concentration.
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