Environ Monit Assess (2015) 187:276 DOI 10.1007/s10661-015-4392-y
Resazurin reduction assay, a useful tool for assessment of heavy metal toxicity in acidic conditions Mohammadreza Zare & Mohammad Mehdi Amin & Mahnaz Nikaeen & Bijan Bina & Hamidreza Pourzamani & Ali Fatehizadeh & Ensieh Taheri
Received: 21 August 2014 / Accepted: 1 December 2014 # Springer International Publishing Switzerland 2015
Abstract Almost all bioassays have been designed only for pH levels around 7; however, some toxicant characteristics may be different at lower pH values. In this study, a modified resazurin reduction method was used to evaluate the toxicity of heavy metals and metal plating wastewater on acid-tolerant (AT) and conventional bacteria at the natural and acidic pH conditions. According to our optimized protocol, resazurin was rapidly reduced by both conventional and AT active microorganisms. Considering the 30-min median effective concentration (30 min EC50) values, conventional bacteria were comparatively more resistant than the acid-tolerant bacteria (ATB) in the case of exposure to Cd, Pb, Cr, and Zn, but the reverse case was found for Hg. After an exposure of 30 min, Cr and Hg showed the highest toxicity to ATB (30 min EC50 values were 0.34 and 17.02 μmol/L, respectively), while Zn and Pb had a considerably lower toxicity. The modified resazurin reduction method successfully assessed the impact of metal plating wastewaters on the activities of conventional and AT bacteria. According to the findings where the wastewaters contain heavy metals, wastewater treatment facilities, which are dependent on ATB activity, M. Zare : M. Nikaeen : B. Bina (*) Department of Environmental Health Engineering and Student Research Center, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran e-mail: [email protected] M. Zare : M. M. Amin : M. Nikaeen : H. Pourzamani : A. Fatehizadeh : E. Taheri Environment Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
should use bioassays at acidic pH values for better understanding of the effects of toxicants. Keywords Resazurin . Dehydrogenase enzyme activity . Bioassay . Heavy metals
Introduction Heavy metals are discharged to wastewater treatment plants (WWTP) from many industrial wastes and are common pollutants of sewage, especially where there is input of metal plating industries. Heavy metals are toxic to nearly all of microorganisms at certain concentrations. Since unidentified components may be present in the field samples, appropriate referencing of the sample toxicity via physical and chemical methods may not be practical. As a solution, whole sample analysis using bioindicators was proposed by the U.S. Environment Protection Agency (USEPA) (Chu and Chow 2002). Exposing the test organisms to the field samples can directly reveal the toxicity of any chemical including those which may not be present in the standard test lists. In many of the wastewater treatment systems, such as simultaneous biohydrogen production (Guo et al. 2008), anaerobic digestion of wastewater (Lee et al. 2013), hybrid anaerobic digester system treating municipal wastewater (Weld and Singh 2011), simulated wastewater decolorization process (Isik 2004), and hybrid constructed wetland (Serrano et al. 2011), the most important trophic level in terms of nutrient cycling and energy flow is the acid-tolerant bacteria (ATB) (Choi and Meier
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2001). Therefore, it is essential to include representatives of this trophic level in a test system designed for protecting the WWTP biological processes. Furthermore, the ability to routinely determine the metabolic activity of ATB in a cost-effective and reliable manner would provide a useful means for monitoring the effectiveness of the wastewater treatment process. Wastewater treatment industries have employed a variety of methods to evaluate microbial activity. In this regard, many studies have explored the use of luminescent bacteria. However, these methods are based on limited bacterial species and provide only partial information about the whole community structure of bacteria (Lopez-Roldan et al. 2012). Since most of the bacteria from environmental samples cannot be cultured, determining their number and diversity must involve molecular tools. Particularly, polymerase chain reactiondenaturing gradient gel electrophoresis (PCR–DGGE) is referred to as a rapid and reliable technique for the relative comparison of the different bacterial communities. But as this technique needs sophisticated instruments and is not cost-effective, it cannot routinely be used in small WWTPs (Muyzer et al. 1993). Some studies have focused on assessing the performance of different sensors utilizing enzymes, bacteria, or vertebrate cells (Van der Schalie et al. 2006). Furthermore, some studies have investigated the inhibitory effects of chemicals on bacterial growth using three commercial assay systems, which are based on a luminescent bacterial toxicity assay (ToxAlert®, Microtox®, and LUMIStox®) (Jennings et al. 2001). To date, no study has addressed the effect of industrial effluents on acid-tolerant (AT) microorganisms. Most of the cost-effective and reliable methods for evaluation of wastewater toxicity on microorganisms use protocols that measure the activity of some microbial enzymes, which are engaged in oxidative substrate removal, for instance electron transport system (ETS) dehydrogenase activity (Packard 1985). It has been shown that sludge ETS activity correlates significantly with oxygen uptake rate (Bensaid et al. 2000) and is usually evaluated by determining the reduction of redox dyes which can effectively compete with oxygen atoms as electron acceptors. Many compounds with such characteristics have been investigated; the most common are soluble tetrazolium salts which are colorless and when reduced transform to insoluble colored formazan products (Kim et al. 1994). However, the need for extracting and solubilizing these formazan products makes it
Environ Monit Assess (2015) 187:276
inconvenient to use tetrazolium reduction for quantification of electron transport system (ETS) activity. An alternative approach is using the commercial products which utilize several new generations of tetrazolium salts and which reduce and form soluble formazan products (McCluskey et al. 2005). However, their use in wastewater treatment plants for routine analysis is not cost-effective. In order to develop an easy, rapid, and cost-effective method for monitoring the bacterial activity within a sequence batch reactor (SBR), we have focused on another soluble redox dye, resazurin (7hydroxy-3H-phenoxazin-3-one-10-oxide). Resazurin, also known as Alamar blue (O’Brien et al. 2000), is a non-toxic tetrazolium-based dye which is reduced to a fluorescent compound, resorufin, by microbial ETS enzymes (Fai and Grant 2009). Since it is a non-toxic compound, it can be used to non-destructively check the viability of microorganisms in aqueous and solid environments (Fai and Grant 2009; Byth et al. 2001; Irha and Blinova 2007; McNicholl et al. 2007) To our knowledge, a resazurin-based assay has not been developed to measure the impact of wastewater heavy metals at acidic conditions on ATB within an SBR. The aim of this study was to determine the impact of heavy metal effluents on the ATB within an SBR using a modified and simplified earlier procedure for determination of resazurin reduction by activated sludge (Liu 1986).
Materials and methods Wastewater samples Metal plating wastewater samples were collected from two plating facilities, located in Isfahan, Iran (August 2013). Effluents from these facilities were transported to an SBR plant, where effluent discharge was used for landscape irrigation. The SBR was operated in a 8-h cycle with 6 h of feeding and reaction phase, 1.5 h of settling phase, and 0.5 h of decanting. Dissolved oxygen (DO) level was maintained at 1.0–3 mg/L using a DO controller system. The reactor was run at ambient temperature (15–28 °C). Other operating parameters of the SBR were as follows: flow rate, 900 m3/day; food to microorganism ratio (F/M), 0.15–0.35 g COD/g MLVSS.day; hydraulic retention time (HRT), 1.5–2.5 day; and solid retention time, 8 day.
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The samples were transported in polyethylene containers on ice and stored at 4 °C. Afterwards, initial toxicity tests were established and water characteristics were measured. Analytical methods All chemicals, of highest purity available, were obtained from Sigma Chemical Co., Poole, UK. In total, five heavy metals were separately used in the lethality tests. The water-soluble salts for the 5 metals were K2Cr2O7, ZnCl2, Pb(NO3)2 HgCl2, and CdCl2. Assays were performed at room temperature in disposable borosilicate tubes with constant shaking. To separate the suspended solids, samples were allowed to settle at room temperature for 2 h and then clear supernatants were analyzed for pH, alkalinity, electrical conductivity (EC), color, total suspended solids (TSS), volatile suspended solids (VSS), chemical oxygen demand (COD), and total organic carbon (TOC) contents according to the Standard Methods (American Public Health Association (APHA) et al. 2005). The concentration of metals were determined using a Trace Scan inductively coupled-plasma optical-emission spectrometer (ICP-OES; Thermo-Electron Corporation, Dreieich, Germany). A clear account on the method and reagents used is given by Alonso et al. (2000). Optical density of resazurin and resorufin (10 mg/L) and also metal plating wastewater at different wavelengths were recorded using a Hach’s DR 5000 spectrophotometer. Preparation of bacterial culture Freshly harvested AT and conventional SBR cells were obtained by cultivation of 0.5 cm3 of SBR samples in acidic and neutral pH nutrient broth (pH 3.7 and 7 for AT and conventional bacteria, respectively). For keeping the sludge age constant during the period of the toxicity experiments, the test cultures were transferred every day to a fresh culture medium. A clean sample of bacteria using an aseptic preparation technique and the aid of a centrifuge was prepared for evaluation of the toxicity of heavy metals. Using a centrifuge set at 6000 rpm for 4 min, the broth culture was centrifuged and the supernatant was discarded. The pellets were resuspended in 20 mL of phosphate buffer (pH 3.7 and 7 for AT and conventional bacteria
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respectively) and centrifuged again at 6000 rpm for 10 min. This step was repeated until the supernatant was clear. The pellets were then suspended in 20 mL of sterile phosphate buffer. The optical density of the suspension was recorded at 540 nm, and using appropriate aseptic techniques, serial dilutions were carried out until the optical density was 1.0±0.05. Oxygen consumption by conventional and AT bacteria was measured as previously described (McCluskey et al. 2005) by means of a dissolved oxygen meter (Hach-Lange Model SC100). Briefly, experiments were carried out in a 1-L Erlenmeyer flask containing 500 mL of nutrient broth inoculated to give a primary optical density of 1 ± 0.05. Consumption of oxygen was compared against resazurin reduction in parallel bacterial suspensions incubated in identical conditions. Optimized resazurin reduction assay at acidic condition Resazurin reduction method of Liu (1986) was modified and used to determine the toxicity of the heavy metals and metal plating wastewater at acidic condition. The assay mixture (5000 μL) contained 500 μL phosphate buffer (pH 3.7 and 7 for AT and conventional bacteria, respectively), 500 μL of nutrient broth (×10), 50 μL of resazurin solution at 1 g/L concentration, and distilled water (pH 3.7 and 7 for AT and conventional bacteria, respectively) to a volume of 4000 μL. This volume was increased to 5000 μL by adding 1000 μL of freshly harvested bacteria. As needed, pH values, were adjusted to 3.7 and 7 for AT and conventional bacteria, respectively by adding 1N HCl or 1N NaOH. When required, artificial effluents were used in the assay at concentrations corresponding to those experienced in the field. The reaction was started by adding 1000 mL of freshly harvested AT or conventional cells. In parallel, bacteria-free experiments were set up as controls. The experiments were incubated in the dark at 21 °C on a shaker at 120 rpm. After 0 and 30 min, a 1000-μL sample volume was removed from the assay. After removing the bacteria by vortexing and centrifugation (4 min at 10,000 rpm), resazurin reduction was determined spectrophotometrically at 600 nm. A bacteria-free control assay containing sterile water was used to blank the spectrophotometer. All assays were carried out in triplicate and the means were calculated. In the existence of active bacterial culture, dehydrogenase enzyme activity changes resazurin to reduced
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Resazurin reducon (%) by Liu (1986) method
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100 80 R² = 0.9603 60 40 20 0 0
20
40
60
80
100
Resazurin reducon (%) by opmised method
Fig. 1 Relationship between our optimized protocol and Liu’s (1986) method, using SBR conventional biomass (biomass dry weight, 200–1000 μg/L)
compound resorufin and the color turns from blue to pink. Inactive bacteria cause no change in resazurin color and it stays blue. Therefore, the resazurin solution color can be an indicator of bacterial activity. The results were recorded as the quantity of heavy metals in mg/L required for reducing the growth of bacteria to 2 standard deviations (no observed effect concentration (NOEC)), to 50 % (50 % effective concentration
Hg
R² = 0.9193
Cd
R² = 0.9361
60
Pb
R² = 0.8941
40
Zn
R² = 0.940
20
Cr
R² = 0.977
Hg
R² = 0.9282
Cd
R² = 0.9039
60
Pb
R² = 0.9212
40
Zn
R² = 0.949
20
Cr
R² = 0.8979
Inhibion of dehydrogenas enzyme acvity (%)
100
(a)
80
0 0
100 Inhibion of dehydrogenas enzyme acvity (%)
Fig. 2 Correlation of resazurin reduction assay (inhibition of dehydrogenase enzyme activity) and CFU inhibition assay. Conventional bacteria (a) and ATB (b). Inoculum cell density= 0.300±0.01 at 540 nm
(EC50), and to 100 % (100 % mortality concentration) of the mean growth level of control cultures. These values were calculated by probit analysis using the SPSS ver. 16.0 software. Because resazurin and resorufin are fluorescent just in their anionic form (pH above 7.0), for ATB assay, after exposure to toxic substances, the pH was raised to 7 and then the resazurin reduction was measured. For confirming the efficiency, robustness, and reproducibility of the optimized method, it was compared with the Liu’s (1986) method and Standardpour plate technique (colony forming unit (CFU)/ mL). Pearson’s correlation test was used to examine the significance of the correlation between these methods (p Cd2+ >Pb2+ >Zn2+. According to these results, it can be concluded that individual heavy metals are different in their toxicity degrees to various microbial populations.
Table 3 Results of 30 min EC50, NOEC, and 100 % mortality tests (with 95 % confidence interval) for conventional and acid-tolerant bacteria exposed to metal plating samples Values
Toxic substances
Bacteria groups Conventional
EC50
NOEC
100 % mortality
Acid-tolerant
Sample 1 (mL/L)
6.53 (5.86–7.29)
4.60 (2.86–7.33)
Sample 2 (mL/L)
215.5 (174.5–263.7)
137.7 (105.6–177.9) 35.88 (20.85–53.95)
Conductivity (mS/cm)
41.43 (32.59–51.83)
Sample 1 (mL/L)
1.15 (0.84–1.48)
0.81 (0.10–1.62)
Sample 2 (mL/L)
66.41 (34.27–94.19)
35.07 (14.38–54.10)
Conductivity (mS/cm)
11.41 (4.27–17.11)
9.17 (0.58–17.36)
Sample 1 (mL/L)
36.93 (28.84–51.18)
25.96 (13.15–198.05)
Sample 2 (mL/L)
699.01 (500.08–1311.71)
540.37 (354.28–1283.19)
Conductivity (mS/cm)
154.05 (102.40–381.05)
140.42 (79.11–1579)
The results were recorded as the quantity of heavy metals in milligrams per liter required for reducing the growth of bacteria to 2 standard deviations (no observed effect concentration (NOEC)), to 50 % (50 % effective concentration (EC50), and to 100 % (100 % mortality concentration) of the mean growth level of control cultures. The EC50 values were calculated by probit analysis using the SPSS ver. 16.0 software. Values are mean of triplicate measurements
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100 80
100
(a)
80 60 40 20 0 1
10 100 Electrical conducvity (mS/cm)
(b)
60 40 20 0 0.1
1 10 100 Sample 1 concentraon (mL/L)
Inhibion of dehydrogenase enzyme acvity (%)
Inhibion of dehydrogenase enzyme acvity (%)
Inhibion of dehydrogenase enzyme acvity (%)
Environ Monit Assess (2015) 187:276
100
(c)
80 60 40 20 0 10
100 1000 Sample 2 concentraon (mL/L)
Fig. 9 Representative field samples and electrical conductivity dose–response graphs from experiments for AT (red lines) and conventional bacteria (blue lines). Electrical conductivity (a), sample 1 (b), and sample 2 (c). Each value is the mean of three
replicates with vertical bars representing the standard error of difference between means at each concentration. Maximum-likelihood probit line and 95 % confidence limits are presented
In previous investigations, resazurin dye has been used for assessment of the effects of heavy metals and polyaromatic hydrocarbons (PAHs) on soil (Irha et al. 2003). The method proved to be useable on all soils. Results of our study proved that in addition to soil, resazurin dye can be used for toxicity assessment in aqueous systems.
of SBR biomass. Measurements were made using both the AT and conventional SBR bacteria. The characteristics of the raw wastewater samples are given in Table 2. Since effluents are considered to be toxic at conductivities over 10,000 μS/cm (VillegasNavarro et al. 1999), the conductivity level of both samples, without dilution, was high enough to be toxic for some organisms (8.0 and 13.6 mS/cm for samples 1 and 2, respectively). For that reason, the EC50 for conductivity was determined. But results showed that conductivity cannot be toxic to AT or conventional bacteria in such values. In addition, 10.3 mg/L Rb in the sample was not toxic for the bacteria and in general, this element serves as a satisfactory substitute for potassium (Lester 1958). Considering the results of EC50, NOEC, and 100 % mortality tests for samples 1 and 2 (Table 3), it is revealed that in comparison with conventional bacteria, ATB are more susceptible to toxic effects of metal plating wastewater. Therefore, where the wastewater contains heavy metals, wastewater treatment facilities which are dependent on bacterial activity should not employ acidic conditions that are useful for domination of ATB. Moreover, in the wastewater treatment systems that rely on the ATB (Isik 2004; Guo et al. 2008; Serrano et al. 2011; Lee et al. 2013), the ATB toxicity tests should be performed separately. A separate bioassay with the ATB in line with the
The effect of metal plating wastewaters on the conventional and AT bacterial activity Heavy metals in real wastewater samples do not exist as pure solutions. Therefore, the analysis results presented above do not reflect the actual conditions in the environment. In real situations, wastewaters are complex mixtures and may have unrecognized synergistic effects on biological processes (Dalzell et al. 2002). To minimize potential shock loading of wastewater plants, it would be advantageous to have a simple and rapid biomass activity assay to determine the potential influence of imported industrial waste on the plant processes as a whole. In order to more fully establish the applicability of our modified assay, we used it to determine the effect of two metal plating wastewaters on the metabolic activity
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conventional bacteria can more efficiently highlight the toxicity effects of toxicants. As shown in Fig. 9, compared with sample 2, sample 1 displayed higher toxicity for both conventional and AT bacteria. According to Table 3, sample 1 induced 50 % inhibition in conventional and AT bacteria at dilutions 6.53 and 4.60 mL/L, respectively. These values for sample 2 are 215.5 and 137.7 mL/L, respectively. The toxicity of this sample can be mostly attributed to Cr with a concentration of 143.3 mg/L which is equal to 2754 μmol/L. At EC50 levels of sample 1 for conventional and AT bacteria (215.5 and 137.7 mL/L, respectively), corresponding Cr concentrations would be 593.5 μmol/L (2754 × 0.2155) and 379.2 μmol/L (2754×0.1377). Such high concentrations should have induced more inhibition in bacterial population in sample 2 (according to our results in Table 1, the 30-min EC50 values of Cr for conventional and AT bacteria are 1.32 and 0.34 μmol/L, respectively). This inconsistency may be due to several factors such as concentration of inorganic anions, concentration of chelating agents, and competition from other cations. According to previous study results, the toxicity of heavy metals can be reduced by some natural or synthetic chelating agents (Sterritt and Lester 1980). In addition, bacteria may generate more or less toxic forms of heavy metals through the biotransformation process (Sterritt and Lester 1980).
Conclusion We developed a simple and rapid colorimetric test protocol, in which level of reduction of the resazurin is correlated with respiration rate and bacterial biomass in both neutral and acidic cultures. The results proved that dehydrogenase enzymes in ATB as well as conventional bacteria can transform resazurin to resorufin. According to the results of this study in comparison with conventional bacteria, ATB are more susceptible to toxic effects of metal plating wastewater. Therefore, where the wastewater contains heavy metals, wastewater treatment facilities which are dependent to bacterial activity should not employ acidic conditions that are useful for domination of ATB. Moreover, in the wastewater treatment systems that rely on the ATB, the ATB toxicity tests should be performed separately to show the toxicity effect of toxicants more efficiently.
Environ Monit Assess (2015) 187:276 Acknowledgment The authors would like to thank the Environment Research Center at the Isfahan University of medical sciences, Isfahan, Iran, for funding support (No. 2412-1) during the preparation of this manuscript. This work was also supported by Department of Environmental Health Engineering, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran.
Conflict of interest The authors declare that they have no conflict of interest.
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Resazurin reduction assay, a useful tool for assessment of heavy metal toxicity in acidic conditions Mohammadreza Zare & Mohammad Mehdi Amin & Mahnaz Nikaeen & Bijan Bina & Hamidreza Pourzamani & Ali Fatehizadeh & Ensieh Taheri
Received: 21 August 2014 / Accepted: 1 December 2014 # Springer International Publishing Switzerland 2015
Abstract Almost all bioassays have been designed only for pH levels around 7; however, some toxicant characteristics may be different at lower pH values. In this study, a modified resazurin reduction method was used to evaluate the toxicity of heavy metals and metal plating wastewater on acid-tolerant (AT) and conventional bacteria at the natural and acidic pH conditions. According to our optimized protocol, resazurin was rapidly reduced by both conventional and AT active microorganisms. Considering the 30-min median effective concentration (30 min EC50) values, conventional bacteria were comparatively more resistant than the acid-tolerant bacteria (ATB) in the case of exposure to Cd, Pb, Cr, and Zn, but the reverse case was found for Hg. After an exposure of 30 min, Cr and Hg showed the highest toxicity to ATB (30 min EC50 values were 0.34 and 17.02 μmol/L, respectively), while Zn and Pb had a considerably lower toxicity. The modified resazurin reduction method successfully assessed the impact of metal plating wastewaters on the activities of conventional and AT bacteria. According to the findings where the wastewaters contain heavy metals, wastewater treatment facilities, which are dependent on ATB activity, M. Zare : M. Nikaeen : B. Bina (*) Department of Environmental Health Engineering and Student Research Center, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran e-mail: [email protected] M. Zare : M. M. Amin : M. Nikaeen : H. Pourzamani : A. Fatehizadeh : E. Taheri Environment Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
should use bioassays at acidic pH values for better understanding of the effects of toxicants. Keywords Resazurin . Dehydrogenase enzyme activity . Bioassay . Heavy metals
Introduction Heavy metals are discharged to wastewater treatment plants (WWTP) from many industrial wastes and are common pollutants of sewage, especially where there is input of metal plating industries. Heavy metals are toxic to nearly all of microorganisms at certain concentrations. Since unidentified components may be present in the field samples, appropriate referencing of the sample toxicity via physical and chemical methods may not be practical. As a solution, whole sample analysis using bioindicators was proposed by the U.S. Environment Protection Agency (USEPA) (Chu and Chow 2002). Exposing the test organisms to the field samples can directly reveal the toxicity of any chemical including those which may not be present in the standard test lists. In many of the wastewater treatment systems, such as simultaneous biohydrogen production (Guo et al. 2008), anaerobic digestion of wastewater (Lee et al. 2013), hybrid anaerobic digester system treating municipal wastewater (Weld and Singh 2011), simulated wastewater decolorization process (Isik 2004), and hybrid constructed wetland (Serrano et al. 2011), the most important trophic level in terms of nutrient cycling and energy flow is the acid-tolerant bacteria (ATB) (Choi and Meier
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2001). Therefore, it is essential to include representatives of this trophic level in a test system designed for protecting the WWTP biological processes. Furthermore, the ability to routinely determine the metabolic activity of ATB in a cost-effective and reliable manner would provide a useful means for monitoring the effectiveness of the wastewater treatment process. Wastewater treatment industries have employed a variety of methods to evaluate microbial activity. In this regard, many studies have explored the use of luminescent bacteria. However, these methods are based on limited bacterial species and provide only partial information about the whole community structure of bacteria (Lopez-Roldan et al. 2012). Since most of the bacteria from environmental samples cannot be cultured, determining their number and diversity must involve molecular tools. Particularly, polymerase chain reactiondenaturing gradient gel electrophoresis (PCR–DGGE) is referred to as a rapid and reliable technique for the relative comparison of the different bacterial communities. But as this technique needs sophisticated instruments and is not cost-effective, it cannot routinely be used in small WWTPs (Muyzer et al. 1993). Some studies have focused on assessing the performance of different sensors utilizing enzymes, bacteria, or vertebrate cells (Van der Schalie et al. 2006). Furthermore, some studies have investigated the inhibitory effects of chemicals on bacterial growth using three commercial assay systems, which are based on a luminescent bacterial toxicity assay (ToxAlert®, Microtox®, and LUMIStox®) (Jennings et al. 2001). To date, no study has addressed the effect of industrial effluents on acid-tolerant (AT) microorganisms. Most of the cost-effective and reliable methods for evaluation of wastewater toxicity on microorganisms use protocols that measure the activity of some microbial enzymes, which are engaged in oxidative substrate removal, for instance electron transport system (ETS) dehydrogenase activity (Packard 1985). It has been shown that sludge ETS activity correlates significantly with oxygen uptake rate (Bensaid et al. 2000) and is usually evaluated by determining the reduction of redox dyes which can effectively compete with oxygen atoms as electron acceptors. Many compounds with such characteristics have been investigated; the most common are soluble tetrazolium salts which are colorless and when reduced transform to insoluble colored formazan products (Kim et al. 1994). However, the need for extracting and solubilizing these formazan products makes it
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inconvenient to use tetrazolium reduction for quantification of electron transport system (ETS) activity. An alternative approach is using the commercial products which utilize several new generations of tetrazolium salts and which reduce and form soluble formazan products (McCluskey et al. 2005). However, their use in wastewater treatment plants for routine analysis is not cost-effective. In order to develop an easy, rapid, and cost-effective method for monitoring the bacterial activity within a sequence batch reactor (SBR), we have focused on another soluble redox dye, resazurin (7hydroxy-3H-phenoxazin-3-one-10-oxide). Resazurin, also known as Alamar blue (O’Brien et al. 2000), is a non-toxic tetrazolium-based dye which is reduced to a fluorescent compound, resorufin, by microbial ETS enzymes (Fai and Grant 2009). Since it is a non-toxic compound, it can be used to non-destructively check the viability of microorganisms in aqueous and solid environments (Fai and Grant 2009; Byth et al. 2001; Irha and Blinova 2007; McNicholl et al. 2007) To our knowledge, a resazurin-based assay has not been developed to measure the impact of wastewater heavy metals at acidic conditions on ATB within an SBR. The aim of this study was to determine the impact of heavy metal effluents on the ATB within an SBR using a modified and simplified earlier procedure for determination of resazurin reduction by activated sludge (Liu 1986).
Materials and methods Wastewater samples Metal plating wastewater samples were collected from two plating facilities, located in Isfahan, Iran (August 2013). Effluents from these facilities were transported to an SBR plant, where effluent discharge was used for landscape irrigation. The SBR was operated in a 8-h cycle with 6 h of feeding and reaction phase, 1.5 h of settling phase, and 0.5 h of decanting. Dissolved oxygen (DO) level was maintained at 1.0–3 mg/L using a DO controller system. The reactor was run at ambient temperature (15–28 °C). Other operating parameters of the SBR were as follows: flow rate, 900 m3/day; food to microorganism ratio (F/M), 0.15–0.35 g COD/g MLVSS.day; hydraulic retention time (HRT), 1.5–2.5 day; and solid retention time, 8 day.
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The samples were transported in polyethylene containers on ice and stored at 4 °C. Afterwards, initial toxicity tests were established and water characteristics were measured. Analytical methods All chemicals, of highest purity available, were obtained from Sigma Chemical Co., Poole, UK. In total, five heavy metals were separately used in the lethality tests. The water-soluble salts for the 5 metals were K2Cr2O7, ZnCl2, Pb(NO3)2 HgCl2, and CdCl2. Assays were performed at room temperature in disposable borosilicate tubes with constant shaking. To separate the suspended solids, samples were allowed to settle at room temperature for 2 h and then clear supernatants were analyzed for pH, alkalinity, electrical conductivity (EC), color, total suspended solids (TSS), volatile suspended solids (VSS), chemical oxygen demand (COD), and total organic carbon (TOC) contents according to the Standard Methods (American Public Health Association (APHA) et al. 2005). The concentration of metals were determined using a Trace Scan inductively coupled-plasma optical-emission spectrometer (ICP-OES; Thermo-Electron Corporation, Dreieich, Germany). A clear account on the method and reagents used is given by Alonso et al. (2000). Optical density of resazurin and resorufin (10 mg/L) and also metal plating wastewater at different wavelengths were recorded using a Hach’s DR 5000 spectrophotometer. Preparation of bacterial culture Freshly harvested AT and conventional SBR cells were obtained by cultivation of 0.5 cm3 of SBR samples in acidic and neutral pH nutrient broth (pH 3.7 and 7 for AT and conventional bacteria, respectively). For keeping the sludge age constant during the period of the toxicity experiments, the test cultures were transferred every day to a fresh culture medium. A clean sample of bacteria using an aseptic preparation technique and the aid of a centrifuge was prepared for evaluation of the toxicity of heavy metals. Using a centrifuge set at 6000 rpm for 4 min, the broth culture was centrifuged and the supernatant was discarded. The pellets were resuspended in 20 mL of phosphate buffer (pH 3.7 and 7 for AT and conventional bacteria
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respectively) and centrifuged again at 6000 rpm for 10 min. This step was repeated until the supernatant was clear. The pellets were then suspended in 20 mL of sterile phosphate buffer. The optical density of the suspension was recorded at 540 nm, and using appropriate aseptic techniques, serial dilutions were carried out until the optical density was 1.0±0.05. Oxygen consumption by conventional and AT bacteria was measured as previously described (McCluskey et al. 2005) by means of a dissolved oxygen meter (Hach-Lange Model SC100). Briefly, experiments were carried out in a 1-L Erlenmeyer flask containing 500 mL of nutrient broth inoculated to give a primary optical density of 1 ± 0.05. Consumption of oxygen was compared against resazurin reduction in parallel bacterial suspensions incubated in identical conditions. Optimized resazurin reduction assay at acidic condition Resazurin reduction method of Liu (1986) was modified and used to determine the toxicity of the heavy metals and metal plating wastewater at acidic condition. The assay mixture (5000 μL) contained 500 μL phosphate buffer (pH 3.7 and 7 for AT and conventional bacteria, respectively), 500 μL of nutrient broth (×10), 50 μL of resazurin solution at 1 g/L concentration, and distilled water (pH 3.7 and 7 for AT and conventional bacteria, respectively) to a volume of 4000 μL. This volume was increased to 5000 μL by adding 1000 μL of freshly harvested bacteria. As needed, pH values, were adjusted to 3.7 and 7 for AT and conventional bacteria, respectively by adding 1N HCl or 1N NaOH. When required, artificial effluents were used in the assay at concentrations corresponding to those experienced in the field. The reaction was started by adding 1000 mL of freshly harvested AT or conventional cells. In parallel, bacteria-free experiments were set up as controls. The experiments were incubated in the dark at 21 °C on a shaker at 120 rpm. After 0 and 30 min, a 1000-μL sample volume was removed from the assay. After removing the bacteria by vortexing and centrifugation (4 min at 10,000 rpm), resazurin reduction was determined spectrophotometrically at 600 nm. A bacteria-free control assay containing sterile water was used to blank the spectrophotometer. All assays were carried out in triplicate and the means were calculated. In the existence of active bacterial culture, dehydrogenase enzyme activity changes resazurin to reduced
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Resazurin reducon (%) by Liu (1986) method
276
100 80 R² = 0.9603 60 40 20 0 0
20
40
60
80
100
Resazurin reducon (%) by opmised method
Fig. 1 Relationship between our optimized protocol and Liu’s (1986) method, using SBR conventional biomass (biomass dry weight, 200–1000 μg/L)
compound resorufin and the color turns from blue to pink. Inactive bacteria cause no change in resazurin color and it stays blue. Therefore, the resazurin solution color can be an indicator of bacterial activity. The results were recorded as the quantity of heavy metals in mg/L required for reducing the growth of bacteria to 2 standard deviations (no observed effect concentration (NOEC)), to 50 % (50 % effective concentration
Hg
R² = 0.9193
Cd
R² = 0.9361
60
Pb
R² = 0.8941
40
Zn
R² = 0.940
20
Cr
R² = 0.977
Hg
R² = 0.9282
Cd
R² = 0.9039
60
Pb
R² = 0.9212
40
Zn
R² = 0.949
20
Cr
R² = 0.8979
Inhibion of dehydrogenas enzyme acvity (%)
100
(a)
80
0 0
100 Inhibion of dehydrogenas enzyme acvity (%)
Fig. 2 Correlation of resazurin reduction assay (inhibition of dehydrogenase enzyme activity) and CFU inhibition assay. Conventional bacteria (a) and ATB (b). Inoculum cell density= 0.300±0.01 at 540 nm
(EC50), and to 100 % (100 % mortality concentration) of the mean growth level of control cultures. These values were calculated by probit analysis using the SPSS ver. 16.0 software. Because resazurin and resorufin are fluorescent just in their anionic form (pH above 7.0), for ATB assay, after exposure to toxic substances, the pH was raised to 7 and then the resazurin reduction was measured. For confirming the efficiency, robustness, and reproducibility of the optimized method, it was compared with the Liu’s (1986) method and Standardpour plate technique (colony forming unit (CFU)/ mL). Pearson’s correlation test was used to examine the significance of the correlation between these methods (p Cd2+ >Pb2+ >Zn2+. According to these results, it can be concluded that individual heavy metals are different in their toxicity degrees to various microbial populations.
Table 3 Results of 30 min EC50, NOEC, and 100 % mortality tests (with 95 % confidence interval) for conventional and acid-tolerant bacteria exposed to metal plating samples Values
Toxic substances
Bacteria groups Conventional
EC50
NOEC
100 % mortality
Acid-tolerant
Sample 1 (mL/L)
6.53 (5.86–7.29)
4.60 (2.86–7.33)
Sample 2 (mL/L)
215.5 (174.5–263.7)
137.7 (105.6–177.9) 35.88 (20.85–53.95)
Conductivity (mS/cm)
41.43 (32.59–51.83)
Sample 1 (mL/L)
1.15 (0.84–1.48)
0.81 (0.10–1.62)
Sample 2 (mL/L)
66.41 (34.27–94.19)
35.07 (14.38–54.10)
Conductivity (mS/cm)
11.41 (4.27–17.11)
9.17 (0.58–17.36)
Sample 1 (mL/L)
36.93 (28.84–51.18)
25.96 (13.15–198.05)
Sample 2 (mL/L)
699.01 (500.08–1311.71)
540.37 (354.28–1283.19)
Conductivity (mS/cm)
154.05 (102.40–381.05)
140.42 (79.11–1579)
The results were recorded as the quantity of heavy metals in milligrams per liter required for reducing the growth of bacteria to 2 standard deviations (no observed effect concentration (NOEC)), to 50 % (50 % effective concentration (EC50), and to 100 % (100 % mortality concentration) of the mean growth level of control cultures. The EC50 values were calculated by probit analysis using the SPSS ver. 16.0 software. Values are mean of triplicate measurements
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100 80
100
(a)
80 60 40 20 0 1
10 100 Electrical conducvity (mS/cm)
(b)
60 40 20 0 0.1
1 10 100 Sample 1 concentraon (mL/L)
Inhibion of dehydrogenase enzyme acvity (%)
Inhibion of dehydrogenase enzyme acvity (%)
Inhibion of dehydrogenase enzyme acvity (%)
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100
(c)
80 60 40 20 0 10
100 1000 Sample 2 concentraon (mL/L)
Fig. 9 Representative field samples and electrical conductivity dose–response graphs from experiments for AT (red lines) and conventional bacteria (blue lines). Electrical conductivity (a), sample 1 (b), and sample 2 (c). Each value is the mean of three
replicates with vertical bars representing the standard error of difference between means at each concentration. Maximum-likelihood probit line and 95 % confidence limits are presented
In previous investigations, resazurin dye has been used for assessment of the effects of heavy metals and polyaromatic hydrocarbons (PAHs) on soil (Irha et al. 2003). The method proved to be useable on all soils. Results of our study proved that in addition to soil, resazurin dye can be used for toxicity assessment in aqueous systems.
of SBR biomass. Measurements were made using both the AT and conventional SBR bacteria. The characteristics of the raw wastewater samples are given in Table 2. Since effluents are considered to be toxic at conductivities over 10,000 μS/cm (VillegasNavarro et al. 1999), the conductivity level of both samples, without dilution, was high enough to be toxic for some organisms (8.0 and 13.6 mS/cm for samples 1 and 2, respectively). For that reason, the EC50 for conductivity was determined. But results showed that conductivity cannot be toxic to AT or conventional bacteria in such values. In addition, 10.3 mg/L Rb in the sample was not toxic for the bacteria and in general, this element serves as a satisfactory substitute for potassium (Lester 1958). Considering the results of EC50, NOEC, and 100 % mortality tests for samples 1 and 2 (Table 3), it is revealed that in comparison with conventional bacteria, ATB are more susceptible to toxic effects of metal plating wastewater. Therefore, where the wastewater contains heavy metals, wastewater treatment facilities which are dependent on bacterial activity should not employ acidic conditions that are useful for domination of ATB. Moreover, in the wastewater treatment systems that rely on the ATB (Isik 2004; Guo et al. 2008; Serrano et al. 2011; Lee et al. 2013), the ATB toxicity tests should be performed separately. A separate bioassay with the ATB in line with the
The effect of metal plating wastewaters on the conventional and AT bacterial activity Heavy metals in real wastewater samples do not exist as pure solutions. Therefore, the analysis results presented above do not reflect the actual conditions in the environment. In real situations, wastewaters are complex mixtures and may have unrecognized synergistic effects on biological processes (Dalzell et al. 2002). To minimize potential shock loading of wastewater plants, it would be advantageous to have a simple and rapid biomass activity assay to determine the potential influence of imported industrial waste on the plant processes as a whole. In order to more fully establish the applicability of our modified assay, we used it to determine the effect of two metal plating wastewaters on the metabolic activity
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conventional bacteria can more efficiently highlight the toxicity effects of toxicants. As shown in Fig. 9, compared with sample 2, sample 1 displayed higher toxicity for both conventional and AT bacteria. According to Table 3, sample 1 induced 50 % inhibition in conventional and AT bacteria at dilutions 6.53 and 4.60 mL/L, respectively. These values for sample 2 are 215.5 and 137.7 mL/L, respectively. The toxicity of this sample can be mostly attributed to Cr with a concentration of 143.3 mg/L which is equal to 2754 μmol/L. At EC50 levels of sample 1 for conventional and AT bacteria (215.5 and 137.7 mL/L, respectively), corresponding Cr concentrations would be 593.5 μmol/L (2754 × 0.2155) and 379.2 μmol/L (2754×0.1377). Such high concentrations should have induced more inhibition in bacterial population in sample 2 (according to our results in Table 1, the 30-min EC50 values of Cr for conventional and AT bacteria are 1.32 and 0.34 μmol/L, respectively). This inconsistency may be due to several factors such as concentration of inorganic anions, concentration of chelating agents, and competition from other cations. According to previous study results, the toxicity of heavy metals can be reduced by some natural or synthetic chelating agents (Sterritt and Lester 1980). In addition, bacteria may generate more or less toxic forms of heavy metals through the biotransformation process (Sterritt and Lester 1980).
Conclusion We developed a simple and rapid colorimetric test protocol, in which level of reduction of the resazurin is correlated with respiration rate and bacterial biomass in both neutral and acidic cultures. The results proved that dehydrogenase enzymes in ATB as well as conventional bacteria can transform resazurin to resorufin. According to the results of this study in comparison with conventional bacteria, ATB are more susceptible to toxic effects of metal plating wastewater. Therefore, where the wastewater contains heavy metals, wastewater treatment facilities which are dependent to bacterial activity should not employ acidic conditions that are useful for domination of ATB. Moreover, in the wastewater treatment systems that rely on the ATB, the ATB toxicity tests should be performed separately to show the toxicity effect of toxicants more efficiently.
Environ Monit Assess (2015) 187:276 Acknowledgment The authors would like to thank the Environment Research Center at the Isfahan University of medical sciences, Isfahan, Iran, for funding support (No. 2412-1) during the preparation of this manuscript. This work was also supported by Department of Environmental Health Engineering, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran.
Conflict of interest The authors declare that they have no conflict of interest.
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