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Effect of organic toxicants on the activity of denitrifying granular sludge a
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Zonghe Zhang , Ping Zheng , Wei Li , Ru Wang & Abbas Ghulam
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Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, People's Republic of China Accepted author version posted online: 15 Sep 2014.Published online: 25 Sep 2014.
Click for updates To cite this article: Zonghe Zhang, Ping Zheng, Wei Li, Ru Wang & Abbas Ghulam (2015) Effect of organic toxicants on the activity of denitrifying granular sludge, Environmental Technology, 36:6, 699-705, DOI: 10.1080/09593330.2014.959065 To link to this article: http://dx.doi.org/10.1080/09593330.2014.959065
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Environmental Technology, 2015 Vol. 36, No. 6, 699–705, http://dx.doi.org/10.1080/09593330.2014.959065
Effect of organic toxicants on the activity of denitrifying granular sludge Zonghe Zhang, Ping Zheng ∗ , Wei Li, Ru Wang and Abbas Ghulam Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, People’s Republic of China
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(Received 27 March 2014; final version received 22 August 2014 ) Denitrification plays a key role in the biological nitrogen removal from the wastewater using granular sludge as the integral part of a high-rate denitrification technology. It is helpful to evaluate the effect of typical organic toxicants on the activity of denitrifying granular sludge for the application of denitrification technology. In this study, four typical organic toxicants, namely, penicillin, chloramphenicol, 2,4-dinitrophenol and polymyxin B sulphate were used to assess the effect of organic toxicants on the activity of denitrifying granular sludge. The results of individual toxicity indicated that penicillin, chloramphenicol and 2,4-dinitrophenol had significant inhibition, whose half-inhibitory concentrations were 0.534, 0.162 and 0.474 g/L with respective inhibitory magnitudes of 90.79%/(g/L), 282.5%/(g/L) and 138.83%/(g/L). Polymyxin B sulphate showed no significant inhibition. The results of combined toxicity indicated that the binary mixture of penicillin and chloramphenicol had an antagonistic effect, both the binary mixture of penicillin and 2,4-dinitrophenol and the binary mixture of chloramphenicol and 2,4-dinitrophenol had additive effects. The ternary mixture of penicillin, chloramphenicol and 2,4-dinitrophenol had a partial additive effect. Keywords: organic toxicant; denitrifying granular sludge; specific activity; toxicity assessment
1. Introduction Chemical and pharmaceutical industries are the economic mainstay in P.R. China, but they contaminate the environment seriously.[1,2] The treatment of wastewater from such industries is difficult due to the complex composition, high concentration and toxic ingredients.[3–5] In chemical and pharmaceutical industries, nitrogen compounds are commonly used as raw materials which necessitate the removal of nitrogenous compounds from the wastewater to meet the emission standards. So far, the most economical method for high-nitrogen wastewater is the biological treatment which incorporates denitrification as an integral part. However, the development and application of denitrification process face a serious challenge due to the presence of high nitrogenous and toxic compounds in the industrial wastewaters.[6] Therefore, it is of great significance and beneficial to investigate the effect of organic toxicants on the activity of denitrifying granular sludge for the further development of denitrification technology. The granular sludge bed reactor is a type of highrate reactor [7] whose granular sludge provides a unique micro-ecology and a good settling property. The functional bacteria in the form of granular sludge are primarily responsible for the high-rate denitrification process in the reactor.[8] Therefore, the assessment of effect of organic toxicants on the activity of granular sludge can indicate the suitability of high-rate denitrification reactor. So far,
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
however, there is almost no information available on the effect of organic toxicants on the activity of denitrifying granular sludge, let alone on the combined effect of organic toxicants on the activity of denitrifying granular sludge. Biological denitrification refers to the process in which nitrate or nitrite is reduced to nitrogen oxides or dinitrogen by the denitrifying microorganisms.[9] It was catalysed by (1) nitrate reductase, (2) nitrite reductase, (3) nitric oxide reductase and (4) nitrous oxide reductase, respectively. The activity of denitrifying granular sludge largely depends on the activity of four kinds of denitrifying enzymes in the functional bacteria. The reaction pathway is as follows: (1)+2e
(2)+e
(3)+e
(4)+e
− NO− 3 −→ NO2 −→ NO −→ N2 O −→ N2 .
Penicillin, chloramphenicol, 2,4-dinitrophenol and polymyxin B sulphate are four typical organic toxicants with distinct toxicological mechanism, functioning in different target organs, which widely exist in various kinds of chemical engineering, pharmaceutical and farm wastewater. Penicillin inhibits the synthesis of bacterial cellwall, chloramphenicol retards the synthesis of protein by means of the 50S subunit of 70S ribosome, 2,4-dinitrophenol is an uncoupler of oxidative phosphorylation, reducing the synthesis of ATP, and polymyxin B sulphate can destroy the cell membrane integrity. In this study, the four typical
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organic toxicants are used as test materials to assess the effect of organic toxicants on denitrifying granular sludge so as to lay a technical basis for the application of a high-rate denitrification reactor.
2. Materials and methods 2.1. Experimental materials The seeding sludge used in the experiment was taken from the high-rate denitrification reactor, which could achieve a volumetric removal rate of 35 kg N/(m3 d). In the denitrification reaction, methanol served as the electron donor and nitrate served as the electron acceptor. As the reactor was under steady state, the concentrations of influent nitrate NO− 3 − N and COD were 1000 and 5000 mg/L, respectively. The suspended solids (SS) and the volatile suspended solids (VSS) of the denitrifying granular sludge were 98.95 and 49.28 g/L, respectively. And the average diameter of the denitrifying granular sludge was 2.39 ± 0.05 mm.[10] The original seeding sludge of the high-rate denitrifying reactor was anaerobic granular sludge that was taken from a large-scale internal circulation (IC) anaerobic reactor located in Zhejiang, China. The high-rate denitrifying reactor has been operated continuously for more than two years. Inorganic reagents used in the experiment were of analytical grade; penicillin, chloramphenicol and 2,4dinitrophenol were purchased from Biological Engineering (Shanghai) Co., Ltd.; and polymyxin B sulphate was purchased from Aladdin Reagent Company.
2.2. Specific activity test Determination of the specific activity of granular sludge was conducted in 65 ml serum bottles. Firstly, 6 ml of the seeding sludge and 6 ml of the culture medium [10] were added into the serum bottle, respectively. Then, 48 ml of nitrate solution was added to make 60 ml of the reaction solution. The nitrate concentrations NO− 3 −N in the reaction solution were 10, 100, 600, 1000, 2000, 3000 and 5000 mg/L, respectively. After addition of the reaction solution, the serum bottle was purged with pure nitrogen, and it was then sealed with a rubber stopper. Three parallel tests were set up in the experiment. The serum bottles were cultivated on the shaker at a temperature of 30°C and at a speed of 120 rev/min. Along with the regular sampling, the concentration of nitrate nitrogen was measured, and the conversion rate of nitrate nitrogen was calculated from the data. Then the VSS of granular sludge was measured, and the specific activity of granular sludge was calculated. Using the Monod equation, the maximum specific activity of granular sludge and the nitrate halfsaturation concentration (Ks) were determined by fitting the data.
2.3.
Individual toxicity test
Individual toxicity test was similar to the specific activity test. The nitrate half-saturation concentration (Ks) determined beforehand served as the substrate concentration in the toxicity test. Three parallel tests were carried out. Each toxicant had several concentrations and each toxicant concentration had three serum bottles for parallel tests. The total volume of reaction solution in a serum bottle was 60 ml and the sampling amount for analysis was 1 ml. The specific activity of granular sludge was separately calculated for the four organic toxicants, and the result was compared with that of the control group (the specific activity of granular sludge under a non-toxic condition), and the relative specific activity of granular sludge was determined. Half-inhibitory concentration (30 min-IC50 ) and inhibitory magnitude were determined by using linear regressions.
2.4.
Combined toxicity test
The combined toxicity test was carried out by mixing the toxicants with equivalent effect.[11] The binary and ternary mixtures were prepared based on the half-inhibitory concentration, and they were used as organic toxicants in the combined toxicity test. The combined toxicity was evaluated by an R-value method,[12] toxic unit method (TU) [12,13] and mixed toxicity index (M TI ) [14,15] method. The mass concentration ratio of the components in the mixture was calculated according to the following equation: χi =
IC50(i) , IC50(a) + IC50(b) + · · · + IC50(i) + · · · + IC50(n) (1)
where χi is the mass concentration ratio of the components in the mixture, χi = 1; IC50(i) is the IC50 value of the component. The expected half-inhibitory concentration of the mixture can be calculated by using the following equation: 1 IC50(E)
=
χi IC50(i)
(i = a, b, . . . n),
(2)
where IC50(E) is the expected IC50 value of various mixtures. The 30 min-IC50(E) of each mixture can be calculated by the 30 min-IC50 of individual toxicant, and then the R value can be calculated by using the following equation: R=
IC50(E) , IC50
(3)
where IC50 is the measured half-inhibitory concentration of each mixture. TU was first proposed by Sprague and Ramsay,[12] and was then developed by Marking and Dawson.[13] It can be
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Environmental Technology Table 1.
The evaluation indexes for different types of combined effects.
Combined effects R M M TI
Synergistic effects
Additive effects
Independent effects
Antagonistic effects
Partial additive effects
R > 2.5 M 1
0.4 < R < 2.5 M =1 M TI = 1
M = M0 M TI = 0
R < 0.4 M > M0 M TI < 0
M0 > M > 1 0 < M TI < 1
calculated by the following equation:
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TU =
Ci , IC50i
(4)
where Ci is the concentration of components in the mixture, IC50i is the IC50 value of each component. For a mixture: M=
n i
TUi =
C1 C2 Cn + + ··· + . IC501 IC502 IC50n
(5)
M . (6) (TUi )max Kˇonemann [14] first used the mixed toxicity index (M TI ) to evaluate the combined toxicity of various mixtures to fish. M TI is defined as M0 =
MTI = 1 −
log M . log M0
(7)
The meaning of M and M 0 are the same as the above. Chen et al. [15] modified the method and applied it to study the toxicity of chemicals to bacteria. If each component in the mixture had equal toxicity, then: MTI = 1 −
log M , log n
Figure 1. The specific activity of granular sludge under non– toxic conditions.
(8)
where n is the number of components in the mixture. The evaluation indexes are given in Table 1. 2.5. Analytical methods The determination of nitrate concentration (NO− 3 − N): Use a syringe to take 1 ml of the sample from 65 ml serum bottle; use UV spectrophotometry to determine A220 and A275 value of the sample at wavelengths of 220 and 275 nm; nitrate nitrogen content of the sample was calculated based on A = A220 − 2 A275 and the standard curve. And the determination of SS and VSS was performed according to the standard methods (APHA, 2005).[16] 3. Results and discussion 3.1. The specific activity of granular sludge under non-toxic condition To determine the nitrate concentration (NO− 3 − N) in the toxicity test, the activity of denitrifying granular sludge
was determined beforehand under the non-toxic condition, and the results are shown in Figure 1. When the nitrate concentration was below 500 mg/L, the nitrate conversion rate increased linearly, and the reaction was close to the first-order reaction. When the nitrate concentration was in the range of 500 and 4500 mg/L, the curve was in a non-linear mode, and it tended to be the mixed order reaction. When the nitrate concentration was higher than 4500 mg/L, the curve flattened, that is, it approached the zero-order reaction. If the experimental data were fit by the Monod equation, the following equation Y = 101.47X /(1001.64 + X ) (R2 = 0.972) was obtained. From the established equation, the maximum nitrate conversion rate (Rmax ) was determined as 101.47 mg/(gVSS) /h and the half-saturation concentration (Ks) as 1001.64 mg/L.
3.2.
Effect of individual toxicity on activity of denitrifying granular sludge To ascertain the effect of four typical organic toxicants on specific activity of denitrifying granular sludge, the individual toxicity test was carried out at the nitrate half-saturation concentration, and the results are shown in Figure 2. The inhibition rate of relative specific activity of denitrifying granular sludge (half-saturation rate under non-toxic condition was taken as the reference) was positively correlated with the concentration
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Figure 2. Individual toxicity to activity of denitrifying granular sludge.
of four organic toxicants. After the data were fit linearly, the regression equation and correlation coefficients were obtained and they are given in Table 2. The halfinhibitory concentrations of penicillin, chloramphenicol, 2,4-dinitrophenol and polymyxin B sulphate were 0.534, 0.162, 0.474 and 0.920 g/L, respectively, and the inhibitory magnitudes were 90.79%/(g/L), 282.5%/(g/L), 138.83%/(g/L) and 26.304%/(g/L), respectively. The order of toxicity was as follows: chloramphenicol > 2,4dinitrophenol > penicillin > polymyxin B sulphate. According to the literatures, chloramphenicol inhibited the activity of denitrifying bacteria mainly by means of the 50S subunit of 70S ribosome, which prevented the receptor of ribosome from binding to the acid terminus of aminoacyl-tRNA, and thereby retarded the synthesis of protein (which is the main component of the enzyme).[17] Since denitrification was catalysed by the denitrifying enzymes, the inhibition of chloramphenicol to the synthesis of enzyme reduced the denitrification rate of granular sludge. It has been reported that the chloramphenicol half-inhibitory concentration to luminescent bacteria was 160 μg/L,[18] which was significantly lower than that to denitrifying granular sludge (0.162 g/L). The reason might be related to the prevention of functional bacteria in the outer layer to those in the inner layer of granular sludge.
Table 2.
It is well known that 2,4-dinitrophenol is an uncoupler of oxidative phosphorylation. It diffuses through the cell membrane and carries protons to the opposite of biomembrane; as a result, eliminating the proton concentration gradient across the biomembrane and reducing the synthesis of ATP.[19] Since the transmembrane transport of substrates (nitrate and methanol) required energy, the inhibition of 2,4-dinitrophenol to the synthesis of ATP slowed down the transmembrane transport of substrates, and in turn it reduced the denitrification rate of granular sludge. It has been reported that the 2,4-dinitrophenol half-inhibitory concentration to luminescent bacteria was 0.098 g/L,[20] which was significantly lower than that to denitrifying granular sludge (0.474 g/L). The reason might also be related to the prevention of functional bacteria in the outer layer to those in the inner layer of granular sludge. Penicillin is an inhibitor to the synthesis of bacterial cell wall. Because its structure is similar to the peptide chains of the cell wall, penicillin can bind to glycopeptide transpeptidase, stopping the synthesis of cell wall and leading to the death of denitrifying bacteria.[21] Since penicillin kills bacteria by stopping the synthesis of the cell wall, it is only effective to the growing bacteria. It is worth mentioning that the denitrifying bacteria in granular sludge involved both growing and mature bacteria. So penicillin could not cause complete inhibition to the denitrification of granular sludge. However, as reported in the literature, a prolonged time of toxicity test resulted in a tremendous change of IC50 values.[22] Polymyxin B sulphate is a destroyer of cell membrane integrity, which leads to the dysfunction and death of cell. However, the molecular weight of polymyxin B sulphate is large (1385.61), and it can enter gram-positive bacteria because of the thick cell wall. Protected by the outer layer of granular sludge, the denitrifying activity of granular sludge was hardly affected by polymyxin B sulphate in the toxicity test. Hence, polymyxin B sulphate was not included in the subsequent combined toxicity tests. Compared with acute toxicity with suspended bacteria as the test target, the half-inhibitory concentration with granular sludge as the test target was significantly higher. The fact might be ascribed to the diffusion barrier of granular sludge originated from the functional bacteria and their
Individual toxicity of organic toxicants.
Organic toxicant Penicillin Chloramphenicol 2,4-Dinitrophenol Polymyxin B sulphate
Linear regression equation Y Y Y Y
= = = =
− 90.79X + 98.5 − 282.5X + 95.713 − 138.83 + 115.8 − 26.304 + 74.205
R2
Inhibitory magnitude %/(g/L)
IC50 g/L
0.9865 0.9725 0.9726 0.8678
90.79 282.5 138.83 26.304
0.534 0.162 0.474 0.920
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Environmental Technology Table 4. The evaluation indexes for various mixture. Mixture A
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R M M TI
Figure 3. Combined toxicity to activity of denitrifying granular sludge.
extracellular polymers.[23,24] Hence, it can be concluded that granular sludge tolerated higher concentrations of organic toxicants, and it was helpful for the application of the high-rate denitrification granular sludge bed reactor to the actual wastewater.
3.3.
Effect of combined toxicity on activity of denitrifying granular sludge
To ascertain the combined effects of organic toxicants on the activity of denitrifying granular sludge, the combined toxicity test was carried out on the basis of the individual toxicity test. The binary and ternary mixtures prepared by the method of equivalent mixing were as follows: A (penicillin + chloramphenicol), B (penicillin + 2,4dinitrophenol), C (chloramphenicol + 2,4-dinitrophenol) and D (penicillin + chloramphenicol + 2,4-dinitrophenol). The results are shown in Figure 3. After the data were fit linearly, the regression equation and correlation coefficients were obtained and they are given in Table 3. The half-inhibitory concentrations of A, B, C and D were 0.819, 0.579, 0.327 and 0.528 g/L, respectively, and the inhibitory magnitudes were 42.075%/(g/L), 130.95%/(g/L), 205.04%/(g/L) and 111.6%/(g/L), respectively. The order of toxicity was as follows: C (chloramphenicol + 2,4-dinitrophenol) > D (penicillin + chloramphenicol + 2,4-dinitrophenol) > B (penicillin + 2,4-dinitrophenol) > A (penicillin + chloramphenicol). Table 3.
C
D
0.9 1.1 0.9
1.0 1.0 1.0
0.7 1.4 0.5
The calculated values of evaluation indexes for various mixtures are listed in Table 4. Judged by the combined effects of mixtures A, B, C and D (Table 1), the evaluation results by three methods were in accordance with one another. It could be concluded that the combined effects of mixtures A, B, C and D were antagonistic effects (penicillin + chloramphenicol), additive effects (penicillin + 2,4-dinitrophenol), additive effects (chloramphenicol 2,4-dinitrophenol) and partial additive effects (penicillin + chloramphenicol + 2,4dinitrophenol), respectively. From the toxicological point of view, the combined effects were additive if the toxicants had a similar structure, similar properties and the same target organ or mechanism of action. The combined effects were independent if the toxicants had different pathways and mechanisms of action. The combined effects were synergistic effects if a toxicant promoted the absorption and accumulation of other toxicants or hindered its degradation and excretion in cells. In the binary mixture of penicillin and chloramphenicol, the combined effects were antagonistic because chloramphenicol inhibited the synthesis of protein and caused the denitrifying bacteria to enter the stationary phase, while penicillin was only valid for the growing bacteria, and so the inhibition of chloramphenicol relieved the inhibition of penicillin. In both the binary mixture of penicillin and 2,4dinitrophenol and the binary mixture of chloramphenicol and 2,4-dinitrophenol, the combined effects were additive since the toxicants had different mechanisms and targets of action on denitrifying bacteria. In the ternary mixture of penicillin, chloramphenicol and 2,4-dinitrophenol, the combined effects were partially additive because the binary mixture of penicillin and chloramphenicol had an
Combined toxicity of organic toxicants. Linear regression equation
Mixture A B C D
0.4 2.4 − 0.3
B
Y Y Y Y
= = = =
− 42.075X + 84.472 − 130.95X + 125.85 − 205.04X + 116.95 − 111.6X + 108.94
R2
Inhibitory magnitude %/(g/L)
IC50 g/L
IC50(E) g/L
0.9358 0.947 0.9687 0.977
42.075 130.95 205.04 111.6
0.819 0.579 0.327 0.528
0.348 0.504 0.318 0.39
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antagonistic effect, while the other binary mixtures had additive effects. In real wastewater, many organic toxicants often exist together, which show combined effects. So far, however, there is no information available on the combined effects of various organic toxicants on the activity of denitrifying granular sludge. The measured parameters in this study are helpful in the design, operation and optimization of denitrification processes. Moreover, from the mathematical point of view, the slope of two straight lines may be unequal although the two straight lines share a midpoint (IC50 ). In this article, the slope of regression line was used to explain the inhibitory magnitude of toxicants, which was defined as the inhibition percentage caused by the unit toxicant. The combined application of halfinhibitory concentration and inhibitory magnitude could improve the characterization of toxicant inhibition. The concept of inhibitory magnitude was put forward for the first time here, and hopefully it would be used more and more in the future. 4.
Conclusions (1) The organic toxicants, penicillin, chloramphenicol and 2,4-dinitrophenol had significant inhibition to the specific activity of denitrifying granular sludge at relatively high toxicant concentrations, whose half-inhibitory concentrations were 0.534, 0.162 and 0.474 g/L with respective inhibitory magnitudes of 90.79%/(g/L), 282.5%/(g/L) and 138.83%/(g/L), respectively. Polymyxin B sulphate showed no significant inhibition. The order of toxicity was as follows: chloramphenicol > 2,4nitrophenol > penicillin > polymyxin B sulphate. (2) The organic toxicants revealed different combined effects on the specific activity of denitrifying granular sludge. The binary mixture of penicillin and chloramphenicol had an antagonistic effect. Both the binary mixture of penicillin and 2,4dinitrophenol and the binary mixture of chloramphenicol and 2,4-dinitrophenol had additive effects. The ternary mixture of penicillin, chloramphenicol and 2,4-dinitrophenol had a partial additive effect.
Acknowledgements This research was supported by the National Sci-Tech Plan Projects of China (2013BAD21B04), the Natural Science Foundation of China (51278457) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (J 20120067).
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Effect of organic toxicants on the activity of denitrifying granular sludge a
a
a
a
Zonghe Zhang , Ping Zheng , Wei Li , Ru Wang & Abbas Ghulam
a
a
Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, People's Republic of China Accepted author version posted online: 15 Sep 2014.Published online: 25 Sep 2014.
Click for updates To cite this article: Zonghe Zhang, Ping Zheng, Wei Li, Ru Wang & Abbas Ghulam (2015) Effect of organic toxicants on the activity of denitrifying granular sludge, Environmental Technology, 36:6, 699-705, DOI: 10.1080/09593330.2014.959065 To link to this article: http://dx.doi.org/10.1080/09593330.2014.959065
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Environmental Technology, 2015 Vol. 36, No. 6, 699–705, http://dx.doi.org/10.1080/09593330.2014.959065
Effect of organic toxicants on the activity of denitrifying granular sludge Zonghe Zhang, Ping Zheng ∗ , Wei Li, Ru Wang and Abbas Ghulam Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, People’s Republic of China
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(Received 27 March 2014; final version received 22 August 2014 ) Denitrification plays a key role in the biological nitrogen removal from the wastewater using granular sludge as the integral part of a high-rate denitrification technology. It is helpful to evaluate the effect of typical organic toxicants on the activity of denitrifying granular sludge for the application of denitrification technology. In this study, four typical organic toxicants, namely, penicillin, chloramphenicol, 2,4-dinitrophenol and polymyxin B sulphate were used to assess the effect of organic toxicants on the activity of denitrifying granular sludge. The results of individual toxicity indicated that penicillin, chloramphenicol and 2,4-dinitrophenol had significant inhibition, whose half-inhibitory concentrations were 0.534, 0.162 and 0.474 g/L with respective inhibitory magnitudes of 90.79%/(g/L), 282.5%/(g/L) and 138.83%/(g/L). Polymyxin B sulphate showed no significant inhibition. The results of combined toxicity indicated that the binary mixture of penicillin and chloramphenicol had an antagonistic effect, both the binary mixture of penicillin and 2,4-dinitrophenol and the binary mixture of chloramphenicol and 2,4-dinitrophenol had additive effects. The ternary mixture of penicillin, chloramphenicol and 2,4-dinitrophenol had a partial additive effect. Keywords: organic toxicant; denitrifying granular sludge; specific activity; toxicity assessment
1. Introduction Chemical and pharmaceutical industries are the economic mainstay in P.R. China, but they contaminate the environment seriously.[1,2] The treatment of wastewater from such industries is difficult due to the complex composition, high concentration and toxic ingredients.[3–5] In chemical and pharmaceutical industries, nitrogen compounds are commonly used as raw materials which necessitate the removal of nitrogenous compounds from the wastewater to meet the emission standards. So far, the most economical method for high-nitrogen wastewater is the biological treatment which incorporates denitrification as an integral part. However, the development and application of denitrification process face a serious challenge due to the presence of high nitrogenous and toxic compounds in the industrial wastewaters.[6] Therefore, it is of great significance and beneficial to investigate the effect of organic toxicants on the activity of denitrifying granular sludge for the further development of denitrification technology. The granular sludge bed reactor is a type of highrate reactor [7] whose granular sludge provides a unique micro-ecology and a good settling property. The functional bacteria in the form of granular sludge are primarily responsible for the high-rate denitrification process in the reactor.[8] Therefore, the assessment of effect of organic toxicants on the activity of granular sludge can indicate the suitability of high-rate denitrification reactor. So far,
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
however, there is almost no information available on the effect of organic toxicants on the activity of denitrifying granular sludge, let alone on the combined effect of organic toxicants on the activity of denitrifying granular sludge. Biological denitrification refers to the process in which nitrate or nitrite is reduced to nitrogen oxides or dinitrogen by the denitrifying microorganisms.[9] It was catalysed by (1) nitrate reductase, (2) nitrite reductase, (3) nitric oxide reductase and (4) nitrous oxide reductase, respectively. The activity of denitrifying granular sludge largely depends on the activity of four kinds of denitrifying enzymes in the functional bacteria. The reaction pathway is as follows: (1)+2e
(2)+e
(3)+e
(4)+e
− NO− 3 −→ NO2 −→ NO −→ N2 O −→ N2 .
Penicillin, chloramphenicol, 2,4-dinitrophenol and polymyxin B sulphate are four typical organic toxicants with distinct toxicological mechanism, functioning in different target organs, which widely exist in various kinds of chemical engineering, pharmaceutical and farm wastewater. Penicillin inhibits the synthesis of bacterial cellwall, chloramphenicol retards the synthesis of protein by means of the 50S subunit of 70S ribosome, 2,4-dinitrophenol is an uncoupler of oxidative phosphorylation, reducing the synthesis of ATP, and polymyxin B sulphate can destroy the cell membrane integrity. In this study, the four typical
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organic toxicants are used as test materials to assess the effect of organic toxicants on denitrifying granular sludge so as to lay a technical basis for the application of a high-rate denitrification reactor.
2. Materials and methods 2.1. Experimental materials The seeding sludge used in the experiment was taken from the high-rate denitrification reactor, which could achieve a volumetric removal rate of 35 kg N/(m3 d). In the denitrification reaction, methanol served as the electron donor and nitrate served as the electron acceptor. As the reactor was under steady state, the concentrations of influent nitrate NO− 3 − N and COD were 1000 and 5000 mg/L, respectively. The suspended solids (SS) and the volatile suspended solids (VSS) of the denitrifying granular sludge were 98.95 and 49.28 g/L, respectively. And the average diameter of the denitrifying granular sludge was 2.39 ± 0.05 mm.[10] The original seeding sludge of the high-rate denitrifying reactor was anaerobic granular sludge that was taken from a large-scale internal circulation (IC) anaerobic reactor located in Zhejiang, China. The high-rate denitrifying reactor has been operated continuously for more than two years. Inorganic reagents used in the experiment were of analytical grade; penicillin, chloramphenicol and 2,4dinitrophenol were purchased from Biological Engineering (Shanghai) Co., Ltd.; and polymyxin B sulphate was purchased from Aladdin Reagent Company.
2.2. Specific activity test Determination of the specific activity of granular sludge was conducted in 65 ml serum bottles. Firstly, 6 ml of the seeding sludge and 6 ml of the culture medium [10] were added into the serum bottle, respectively. Then, 48 ml of nitrate solution was added to make 60 ml of the reaction solution. The nitrate concentrations NO− 3 −N in the reaction solution were 10, 100, 600, 1000, 2000, 3000 and 5000 mg/L, respectively. After addition of the reaction solution, the serum bottle was purged with pure nitrogen, and it was then sealed with a rubber stopper. Three parallel tests were set up in the experiment. The serum bottles were cultivated on the shaker at a temperature of 30°C and at a speed of 120 rev/min. Along with the regular sampling, the concentration of nitrate nitrogen was measured, and the conversion rate of nitrate nitrogen was calculated from the data. Then the VSS of granular sludge was measured, and the specific activity of granular sludge was calculated. Using the Monod equation, the maximum specific activity of granular sludge and the nitrate halfsaturation concentration (Ks) were determined by fitting the data.
2.3.
Individual toxicity test
Individual toxicity test was similar to the specific activity test. The nitrate half-saturation concentration (Ks) determined beforehand served as the substrate concentration in the toxicity test. Three parallel tests were carried out. Each toxicant had several concentrations and each toxicant concentration had three serum bottles for parallel tests. The total volume of reaction solution in a serum bottle was 60 ml and the sampling amount for analysis was 1 ml. The specific activity of granular sludge was separately calculated for the four organic toxicants, and the result was compared with that of the control group (the specific activity of granular sludge under a non-toxic condition), and the relative specific activity of granular sludge was determined. Half-inhibitory concentration (30 min-IC50 ) and inhibitory magnitude were determined by using linear regressions.
2.4.
Combined toxicity test
The combined toxicity test was carried out by mixing the toxicants with equivalent effect.[11] The binary and ternary mixtures were prepared based on the half-inhibitory concentration, and they were used as organic toxicants in the combined toxicity test. The combined toxicity was evaluated by an R-value method,[12] toxic unit method (TU) [12,13] and mixed toxicity index (M TI ) [14,15] method. The mass concentration ratio of the components in the mixture was calculated according to the following equation: χi =
IC50(i) , IC50(a) + IC50(b) + · · · + IC50(i) + · · · + IC50(n) (1)
where χi is the mass concentration ratio of the components in the mixture, χi = 1; IC50(i) is the IC50 value of the component. The expected half-inhibitory concentration of the mixture can be calculated by using the following equation: 1 IC50(E)
=
χi IC50(i)
(i = a, b, . . . n),
(2)
where IC50(E) is the expected IC50 value of various mixtures. The 30 min-IC50(E) of each mixture can be calculated by the 30 min-IC50 of individual toxicant, and then the R value can be calculated by using the following equation: R=
IC50(E) , IC50
(3)
where IC50 is the measured half-inhibitory concentration of each mixture. TU was first proposed by Sprague and Ramsay,[12] and was then developed by Marking and Dawson.[13] It can be
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The evaluation indexes for different types of combined effects.
Combined effects R M M TI
Synergistic effects
Additive effects
Independent effects
Antagonistic effects
Partial additive effects
R > 2.5 M 1
0.4 < R < 2.5 M =1 M TI = 1
M = M0 M TI = 0
R < 0.4 M > M0 M TI < 0
M0 > M > 1 0 < M TI < 1
calculated by the following equation:
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TU =
Ci , IC50i
(4)
where Ci is the concentration of components in the mixture, IC50i is the IC50 value of each component. For a mixture: M=
n i
TUi =
C1 C2 Cn + + ··· + . IC501 IC502 IC50n
(5)
M . (6) (TUi )max Kˇonemann [14] first used the mixed toxicity index (M TI ) to evaluate the combined toxicity of various mixtures to fish. M TI is defined as M0 =
MTI = 1 −
log M . log M0
(7)
The meaning of M and M 0 are the same as the above. Chen et al. [15] modified the method and applied it to study the toxicity of chemicals to bacteria. If each component in the mixture had equal toxicity, then: MTI = 1 −
log M , log n
Figure 1. The specific activity of granular sludge under non– toxic conditions.
(8)
where n is the number of components in the mixture. The evaluation indexes are given in Table 1. 2.5. Analytical methods The determination of nitrate concentration (NO− 3 − N): Use a syringe to take 1 ml of the sample from 65 ml serum bottle; use UV spectrophotometry to determine A220 and A275 value of the sample at wavelengths of 220 and 275 nm; nitrate nitrogen content of the sample was calculated based on A = A220 − 2 A275 and the standard curve. And the determination of SS and VSS was performed according to the standard methods (APHA, 2005).[16] 3. Results and discussion 3.1. The specific activity of granular sludge under non-toxic condition To determine the nitrate concentration (NO− 3 − N) in the toxicity test, the activity of denitrifying granular sludge
was determined beforehand under the non-toxic condition, and the results are shown in Figure 1. When the nitrate concentration was below 500 mg/L, the nitrate conversion rate increased linearly, and the reaction was close to the first-order reaction. When the nitrate concentration was in the range of 500 and 4500 mg/L, the curve was in a non-linear mode, and it tended to be the mixed order reaction. When the nitrate concentration was higher than 4500 mg/L, the curve flattened, that is, it approached the zero-order reaction. If the experimental data were fit by the Monod equation, the following equation Y = 101.47X /(1001.64 + X ) (R2 = 0.972) was obtained. From the established equation, the maximum nitrate conversion rate (Rmax ) was determined as 101.47 mg/(gVSS) /h and the half-saturation concentration (Ks) as 1001.64 mg/L.
3.2.
Effect of individual toxicity on activity of denitrifying granular sludge To ascertain the effect of four typical organic toxicants on specific activity of denitrifying granular sludge, the individual toxicity test was carried out at the nitrate half-saturation concentration, and the results are shown in Figure 2. The inhibition rate of relative specific activity of denitrifying granular sludge (half-saturation rate under non-toxic condition was taken as the reference) was positively correlated with the concentration
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Figure 2. Individual toxicity to activity of denitrifying granular sludge.
of four organic toxicants. After the data were fit linearly, the regression equation and correlation coefficients were obtained and they are given in Table 2. The halfinhibitory concentrations of penicillin, chloramphenicol, 2,4-dinitrophenol and polymyxin B sulphate were 0.534, 0.162, 0.474 and 0.920 g/L, respectively, and the inhibitory magnitudes were 90.79%/(g/L), 282.5%/(g/L), 138.83%/(g/L) and 26.304%/(g/L), respectively. The order of toxicity was as follows: chloramphenicol > 2,4dinitrophenol > penicillin > polymyxin B sulphate. According to the literatures, chloramphenicol inhibited the activity of denitrifying bacteria mainly by means of the 50S subunit of 70S ribosome, which prevented the receptor of ribosome from binding to the acid terminus of aminoacyl-tRNA, and thereby retarded the synthesis of protein (which is the main component of the enzyme).[17] Since denitrification was catalysed by the denitrifying enzymes, the inhibition of chloramphenicol to the synthesis of enzyme reduced the denitrification rate of granular sludge. It has been reported that the chloramphenicol half-inhibitory concentration to luminescent bacteria was 160 μg/L,[18] which was significantly lower than that to denitrifying granular sludge (0.162 g/L). The reason might be related to the prevention of functional bacteria in the outer layer to those in the inner layer of granular sludge.
Table 2.
It is well known that 2,4-dinitrophenol is an uncoupler of oxidative phosphorylation. It diffuses through the cell membrane and carries protons to the opposite of biomembrane; as a result, eliminating the proton concentration gradient across the biomembrane and reducing the synthesis of ATP.[19] Since the transmembrane transport of substrates (nitrate and methanol) required energy, the inhibition of 2,4-dinitrophenol to the synthesis of ATP slowed down the transmembrane transport of substrates, and in turn it reduced the denitrification rate of granular sludge. It has been reported that the 2,4-dinitrophenol half-inhibitory concentration to luminescent bacteria was 0.098 g/L,[20] which was significantly lower than that to denitrifying granular sludge (0.474 g/L). The reason might also be related to the prevention of functional bacteria in the outer layer to those in the inner layer of granular sludge. Penicillin is an inhibitor to the synthesis of bacterial cell wall. Because its structure is similar to the peptide chains of the cell wall, penicillin can bind to glycopeptide transpeptidase, stopping the synthesis of cell wall and leading to the death of denitrifying bacteria.[21] Since penicillin kills bacteria by stopping the synthesis of the cell wall, it is only effective to the growing bacteria. It is worth mentioning that the denitrifying bacteria in granular sludge involved both growing and mature bacteria. So penicillin could not cause complete inhibition to the denitrification of granular sludge. However, as reported in the literature, a prolonged time of toxicity test resulted in a tremendous change of IC50 values.[22] Polymyxin B sulphate is a destroyer of cell membrane integrity, which leads to the dysfunction and death of cell. However, the molecular weight of polymyxin B sulphate is large (1385.61), and it can enter gram-positive bacteria because of the thick cell wall. Protected by the outer layer of granular sludge, the denitrifying activity of granular sludge was hardly affected by polymyxin B sulphate in the toxicity test. Hence, polymyxin B sulphate was not included in the subsequent combined toxicity tests. Compared with acute toxicity with suspended bacteria as the test target, the half-inhibitory concentration with granular sludge as the test target was significantly higher. The fact might be ascribed to the diffusion barrier of granular sludge originated from the functional bacteria and their
Individual toxicity of organic toxicants.
Organic toxicant Penicillin Chloramphenicol 2,4-Dinitrophenol Polymyxin B sulphate
Linear regression equation Y Y Y Y
= = = =
− 90.79X + 98.5 − 282.5X + 95.713 − 138.83 + 115.8 − 26.304 + 74.205
R2
Inhibitory magnitude %/(g/L)
IC50 g/L
0.9865 0.9725 0.9726 0.8678
90.79 282.5 138.83 26.304
0.534 0.162 0.474 0.920
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Environmental Technology Table 4. The evaluation indexes for various mixture. Mixture A
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R M M TI
Figure 3. Combined toxicity to activity of denitrifying granular sludge.
extracellular polymers.[23,24] Hence, it can be concluded that granular sludge tolerated higher concentrations of organic toxicants, and it was helpful for the application of the high-rate denitrification granular sludge bed reactor to the actual wastewater.
3.3.
Effect of combined toxicity on activity of denitrifying granular sludge
To ascertain the combined effects of organic toxicants on the activity of denitrifying granular sludge, the combined toxicity test was carried out on the basis of the individual toxicity test. The binary and ternary mixtures prepared by the method of equivalent mixing were as follows: A (penicillin + chloramphenicol), B (penicillin + 2,4dinitrophenol), C (chloramphenicol + 2,4-dinitrophenol) and D (penicillin + chloramphenicol + 2,4-dinitrophenol). The results are shown in Figure 3. After the data were fit linearly, the regression equation and correlation coefficients were obtained and they are given in Table 3. The half-inhibitory concentrations of A, B, C and D were 0.819, 0.579, 0.327 and 0.528 g/L, respectively, and the inhibitory magnitudes were 42.075%/(g/L), 130.95%/(g/L), 205.04%/(g/L) and 111.6%/(g/L), respectively. The order of toxicity was as follows: C (chloramphenicol + 2,4-dinitrophenol) > D (penicillin + chloramphenicol + 2,4-dinitrophenol) > B (penicillin + 2,4-dinitrophenol) > A (penicillin + chloramphenicol). Table 3.
C
D
0.9 1.1 0.9
1.0 1.0 1.0
0.7 1.4 0.5
The calculated values of evaluation indexes for various mixtures are listed in Table 4. Judged by the combined effects of mixtures A, B, C and D (Table 1), the evaluation results by three methods were in accordance with one another. It could be concluded that the combined effects of mixtures A, B, C and D were antagonistic effects (penicillin + chloramphenicol), additive effects (penicillin + 2,4-dinitrophenol), additive effects (chloramphenicol 2,4-dinitrophenol) and partial additive effects (penicillin + chloramphenicol + 2,4dinitrophenol), respectively. From the toxicological point of view, the combined effects were additive if the toxicants had a similar structure, similar properties and the same target organ or mechanism of action. The combined effects were independent if the toxicants had different pathways and mechanisms of action. The combined effects were synergistic effects if a toxicant promoted the absorption and accumulation of other toxicants or hindered its degradation and excretion in cells. In the binary mixture of penicillin and chloramphenicol, the combined effects were antagonistic because chloramphenicol inhibited the synthesis of protein and caused the denitrifying bacteria to enter the stationary phase, while penicillin was only valid for the growing bacteria, and so the inhibition of chloramphenicol relieved the inhibition of penicillin. In both the binary mixture of penicillin and 2,4dinitrophenol and the binary mixture of chloramphenicol and 2,4-dinitrophenol, the combined effects were additive since the toxicants had different mechanisms and targets of action on denitrifying bacteria. In the ternary mixture of penicillin, chloramphenicol and 2,4-dinitrophenol, the combined effects were partially additive because the binary mixture of penicillin and chloramphenicol had an
Combined toxicity of organic toxicants. Linear regression equation
Mixture A B C D
0.4 2.4 − 0.3
B
Y Y Y Y
= = = =
− 42.075X + 84.472 − 130.95X + 125.85 − 205.04X + 116.95 − 111.6X + 108.94
R2
Inhibitory magnitude %/(g/L)
IC50 g/L
IC50(E) g/L
0.9358 0.947 0.9687 0.977
42.075 130.95 205.04 111.6
0.819 0.579 0.327 0.528
0.348 0.504 0.318 0.39
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antagonistic effect, while the other binary mixtures had additive effects. In real wastewater, many organic toxicants often exist together, which show combined effects. So far, however, there is no information available on the combined effects of various organic toxicants on the activity of denitrifying granular sludge. The measured parameters in this study are helpful in the design, operation and optimization of denitrification processes. Moreover, from the mathematical point of view, the slope of two straight lines may be unequal although the two straight lines share a midpoint (IC50 ). In this article, the slope of regression line was used to explain the inhibitory magnitude of toxicants, which was defined as the inhibition percentage caused by the unit toxicant. The combined application of halfinhibitory concentration and inhibitory magnitude could improve the characterization of toxicant inhibition. The concept of inhibitory magnitude was put forward for the first time here, and hopefully it would be used more and more in the future. 4.
Conclusions (1) The organic toxicants, penicillin, chloramphenicol and 2,4-dinitrophenol had significant inhibition to the specific activity of denitrifying granular sludge at relatively high toxicant concentrations, whose half-inhibitory concentrations were 0.534, 0.162 and 0.474 g/L with respective inhibitory magnitudes of 90.79%/(g/L), 282.5%/(g/L) and 138.83%/(g/L), respectively. Polymyxin B sulphate showed no significant inhibition. The order of toxicity was as follows: chloramphenicol > 2,4nitrophenol > penicillin > polymyxin B sulphate. (2) The organic toxicants revealed different combined effects on the specific activity of denitrifying granular sludge. The binary mixture of penicillin and chloramphenicol had an antagonistic effect. Both the binary mixture of penicillin and 2,4dinitrophenol and the binary mixture of chloramphenicol and 2,4-dinitrophenol had additive effects. The ternary mixture of penicillin, chloramphenicol and 2,4-dinitrophenol had a partial additive effect.
Acknowledgements This research was supported by the National Sci-Tech Plan Projects of China (2013BAD21B04), the Natural Science Foundation of China (51278457) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (J 20120067).
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