Article pubs.acs.org/est
Modeling and Application of a Rapid Fluorescence-Based Assay for Biotoxicity in Anaerobic Digestion Jian Lin Chen,† Terry W. J. Steele,*,‡ and David C. Stuckey*,†,§ †
Nanyang Environment & Water Research Institute, Advanced Environmental Biotechnology Centre, Nanyang Technological University, Singapore 637141 ‡ School of Materials Science & Engineering, College of Engineering, Nanyang Technological University, Singapore 637141 § Department of Chemical Engineering, Imperial College London, London SW7 2AZ, U.K. S Supporting Information *
ABSTRACT: The sensitivity of anaerobic digestion metabolism to a wide range of solutes makes it important to be able to monitor toxicants in the feed to anaerobic digesters to optimize their operation. In this study, a rapid fluorescence measurement technique based on resazurin reduction using a microplate reader was developed and applied for the detection of toxicants and/or inhibitors to digesters. A kinetic model was developed to describe the process of resazurin reduced to resorufin, and eventually to dihydroresorufin under anaerobic conditions. By modeling the assay results of resazurin (0.05, 0.1, 0.2, and 0.4 mM) reduction by a pure facultative anaerobic strain, Enterococcus faecalis, and fresh mixed anaerobic sludge, with or without 10 mg L−1 spiked pentachlorophenol (PCP), we found it was clear that the pseudo-first-order rate constant for the reduction of resazurin to resorufin, k1, was a good measure of “toxicity”. With lower biomass density and the optimal resazurin addition (0.1 mM), the toxicity of 10 mg L−1 PCP for E. faecalis and fresh anaerobic sludge was detected in 10 min. By using this model, the toxicity differences among seven chlorophenols to E. faecalis and fresh mixed anaerobic sludge were elucidated within 30 min. The toxicity differences determined by this assay were comparable to toxicity sequences of various chlorophenols reported in the literature. These results suggest that the assay developed in this study not only can quickly detect toxicants for anaerobic digestion but also can efficiently detect the toxicity differences among a variety of similar toxicants.
■
INTRODUCTION Anaerobic digestion breaks down organic waste in the absence of oxygen, producing renewable energy (methane), biofertilizer, and water, and is widely used for treating livestock manure, municipal wastewater solids, food waste, and even high-strength industrial and hospital wastewater. However, a wide range of substances can inhibit anaerobic digestion and cause anaerobic digester upset or failure.1 Therefore, monitoring of toxicants in their feed is critical for keeping anaerobic digesters functioning efficiently. Anaerobic toxicity assays (ATAs)2 have been widely used as a bioassay for monitoring anaerobic digestion for many decades. However, they are time-consuming (12−24 h), and this limits their application when giving advanced warning of toxicants to anaerobic digestion. In addition, many other bioassays have been developed to detect toxicity in anaerobic digestion. For instance, a titration bioassay was proposed for acetoclastic methanogenic activity measurement in fresh anaerobic sludge, but it still took 2 h to identify chloroform inhibition.3 As a time-saving, cost-effective, and simple operation, luminescent bacterial toxicity assays have been widely used for environmental pollution monitoring, and even for anaerobic digestion. However, some luminescent bacterial © XXXX American Chemical Society
strains do not respond to specific chemicals that are toxic to anaerobic digestion, limiting their application when monitoring anaerobic digestion.4 Biosensors, mostly using the respiratory or metabolic functions of microorganisms to detect the inhibition of bioprocesses, are more popular in determining toxicity in anaerobic digestion. The RANTOX biosensor has been developed for monitoring overload and toxicity in anaerobic digestion by using maximal biogas production to detect such incidents.5 However, its slow response time has limited its widespread application because it took 3 h to detect the effect of a step load of 1.07 mg L−1 chloroform on anaerobic sludge.6 To monitor anaerobic digestion toxicity more efficiently, a microbial fuel cell (MFC)-based biosensor has been proposed. However, focusing on the measurement of specific compounds, e.g., volatile fatty acids,7 particularly acetate,8 makes these real-time methods unreliable and slow to respond to toxicants. Shock organic or hydraulic overloads, Received: June 24, 2015 Revised: August 27, 2015 Accepted: October 12, 2015
A
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
subcultured until the middle log phase (∼3 h). An anaerobic reactor was operated at ∼30 °C as the seed for the toxicity test. The reactor was fed weekly with a synthetic medium12 and acetic acid to give a final COD (chemical oxygen demand) of around 2 g L−1. Reduction of Resorufin to Dihydroresorufin under Anaerobic Conditions. Fresh anaerobic sludge (300 μL; volatile suspended solids (VSS), 2.33 g L−1) collected from the anaerobic reactor and subcultured E. faecalis (OD600 = 0.84) described above were transferred into a 96-well microplate with nitrogen gas purging. Resorufin was added to each well, resulting in a concentration of 0.05 mM. The microplate was sealed using sterile film, and triplicate experiments for each test were conducted at 35 °C. The fluorescent signal was read once every 6 min over 3 h by an Infinite M200 Pro microplate reader (TECAN, Singapore) with TECAN i-control software at a λex of 530 nm and a λem of 590 nm with a gain of 30.13 After this, the microplate was exposed to air for 24 h. Then 150 μL of this sludge and bacterial medium exposed to air were taken and mixed with 150 μL of fresh anaerobic sludge or subcultured E. faecalis in a microplate, which was purged by N2 and sealed with a sterile film. Fluorescent signals were read by a microplate reader under the same conditions once every 2 min until the signal plateaued (∼1 h). Resazurin Reduction by E. faecalis and Anaerobic Sludge with and without Spiked PCP. The experimental setup here was one in which appropriate amounts of subcultured bacteria (described above) were mixed with fresh autoclaved BHI broth in a 96-well microplate, resulting in final OD600 values of 0.1, 0.2, and 0.3 (the background OD600 of the BHI broth was 0.05). In one experiment, appropriate amounts of anaerobic sludge were transferred into a 96-well microplate and were diluted to VSS at 1.16 g L−1 by autoclaved deionized (DI) water after it had been collected from the anaerobic reactor. Specific amounts of resazurin were then added to the microplates for a final assay volume of 300 μL/well, yielding final resazurin concentrations of 0.05, 0.1, 0.2, and 0.4 mM. Comparative experiments were set up for both E. faecalis and anaerobic sludge by spiking PCP into the microplates, yielding a final PCP concentration of 10 mg L−1 (parts per million) at the different resazurin concentrations described above. The 96well microplate was sealed immediately with sterile film, and triplicate experiments were conducted for each test at 25 °C. Fluorescence was read once every 2 min for 1 h by the microplate reader under the same conditions described above. Identifying Toxicity Differences among Seven Chlorophenols to E. faecalis and Anaerobic Sludge by the Resazurin Reduction Assay. Appropriate amounts of subcultured bacteria (E. faecalis) were mixed with fresh autoclaved BHI broth in a 96-well microplate to result in a final OD600 of 0.1. Resazurin was also added to the microplate for a final assay volume of 300 μL/well, yielding a final resazurin concentration of 0.1 mM. In addition, seven chlorophenols were also spiked into the microplates, yielding a final concentration of 10 mg L−1, and the microplates were then sealed immediately with sterile film. Triplicate experiments for each test were conducted at 35 °C in a microplate reader, and fluorescence was read every 1 min for 30 min by an Infinite M200 Pro microplate under the same conditions described above. The same experiment with the seven compounds was also conducted with anaerobic sludge, except that the anaerobic sludge was diluted by autoclaved deionized (DI) water in a 1:1 (v:v) ratio.
as well as toxic events, require prompt corrective action to reverse the effects and restore the system’s balance as quickly as possible. Resazurin, which is typically formulated into in vitro toxicology assay kits (i.e., AlamarBlue), can be used as an oxidation−reduction indicator, which results in colorimetric changes and a fluorescent signal in response to intercellular metabolic activity9 based on the reduction of resazurin to resorufin, and finally to dihydroresorufin (Scheme 1). A Scheme 1. Conversion of Resazurin to Resorufin and Dihydroresorufin29,a
At a λex of 530 nm and a λem of 590 nm, resazurin is blue and weakly fluorescent, resorufin is pink and strongly fluorescent, dihydroresorufin is colorless and nonfluorescent.13 a
resazurin reduction assay has been suggested to be a simple, fast, and cheap way to continuously monitor the viability and proliferation of bacterial and eukaryotic cells because of its high water solubility, stability in culture medium, nontoxicity. and permeability through cell membranes.10 However, application of this assay to anaerobic digestion to identify toxicants has not been rigorously investigated. In this study, we investigated the use of a resazurin reduction assay with both a pure culture of facultative bacteria, Enterococcus faecalis, and a mixed anaerobic sludge subjected to spiked toxicants, pentachlorophenol (PCP) and other chlorophenol variants. By modeling the kinetics of resazurin reduction, we showed that this fluorescence measurement not only can detect toxicants but also can detect differences among a variety of similar toxicants.
■
METHODS Chemicals. Resazurin (7-hydroxy-3H-phenoxazin-3-one 10oxide), resorufin (7-hydroxy-3H-phenoxazin-3-one), brain heart infusion (BHI) broth, and seven types of chlorophenols, i.e., pentachlorophenol (PCP), 2-monochlorophenol (2-CP), 3monochlorophenol (3-CP), 4-monochlorophenol (4-CP), 2,3dichlorophenol (2,3-DCP), 2,5-dichlorophenol (2,5-DCP), and 2,4,6-trichlorophenol (2,4,6-TCP), were bought from SigmaAldrich (Singapore). All other reagents and chemicals used were of the highest available purity and were obtained from Sigma-Aldrich. Bacterial Strain and Anaerobic Sludge. It is known that the second largest group of bacteria in an anaerobic digestion community is the class of Bacilli in the domain of Bacteria, of which the most abundant species found in digesters was E. faecalis, which participates in the hydrolysis step of anaerobic digestion and possesses hydrogenase activity in its formate dehydrogenase complex.11 E. faecalis is a facultative bacterium, and all experiments in this study were conducted under anaerobic conditions, making E. faecalis metabolize via fermentation or anaerobic respiration. E. faecalis OG1RF (ATCC 47077) stored at −80 °C in a BHI broth/25% glycerol mixture was gently thawed, and 100 μL was injected anaerobically into 10 mL of N2-sparged, sterile BHI medium. The culture was incubated overnight, and 100 μL was inoculated into an additional 10 mL of BHI medium and B
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Reaction Kinetics of Resazurin Reduction by Living Cells. The reduction of resazurin can be described as10 resazurin (A) + M1 → resorufin (B) + M 2 ↔ dihydroresorufin (C)
where M1 and M2 are metabolites such as NADH, etc. The conversion of resazurin to resorufin and that of resorufin to dihydroresorufin have different pathways, and probably different reaction rates. The conversion of resazurin to resorufin involves cleavage of a N−O bond, while the resorufin to dihydroresorufin reduction is an electron-coupled proton transfer reaction.14 When formed in an aerobic system, dihydroresorufin is immediately oxidized by oxygen, regenerating the resorufin dye.15 However, under anaerobic conditions, we assume that oxidation reactions are negligible,16 and therefore, the reaction can be rewritten as
Figure 1. Reduction of resorufin under anaerobic conditions. Resorufin is irreversibly reduced by anaerobic sludge and E. faecalis under anaerobic conditions but can be regenerated under aerobic conditions.
→(k2) dihydroresorufin (C), in anaerobic systems. Therefore, instead of resazurin, sometimes resorufin may be used as an indicator dye in fluorescence assays as metabolically active cells convert resorufin into nonfluorescent dihydroresorufin.16 Kinetics of Resazurin Reduction by E. faecalis: Effect of Original Resazurin Concentration, Inoculum Density, and Spiked PCP. Figure 2 shows the modeling of resazurin reduction by E. faecalis under different experimental conditions, e.g., bacterial density (OD600), resazurin concentration, and spiked PCP concentration. The simulation results are summarized in Table 1. Most simulation R2 values were greater than 0.9, which is considerably greater than 0.214, the critical R2 value with 30 data points at P = 0.01, suggesting that the model (eq S10) can describe resazurin reduction by E. faecalis quite well. With or without the addition of PCP, the maximal resorufin concentration was around 0.07 mM for addition of 0.1, 0.2, and 0.4 mM resazurin (Figure 2b−d). For the results at an OD600 of 0.1 with addition of 0.2 and 0.4 mM resazurin (Figure 2c,d), if the experimental time had been prolonged, the same maximal resorufin concentration could have been reached. Higher concentrations of resazurin do not seem to result in a higher resorufin concentration; however, 0.05 mM resazurin resulted in resorufin concentrations [∼0.05 mM (Figure 2a)] significantly lower than higher resazurin concentrations. In addition, the inoculum density also had no significant effect on the maximal resorufin concentration; e.g., with the same resazurin concentration, the maximal resorufin concentration was the same for OD600 values of 0.1, 0.2, and 0.3. However, increasing inoculum densities (from an OD600 of 0.1 to 0.2 or 0.3) increased the kinetics of resazurin reduction, making it difficult to determine the effect of addition of PCP on resazurin reduction in the OD600 = 0.2 and 0.3 systems (k1 values summarized in Table 1) with nearly the same tbest‑B. In contrast, at a lower inoculum density (OD600 = 0.1), the addition of 10 mg L−1 PCP clearly slowed the kinetics of resazurin reduction. Therefore, on the basis of this difference in reduction kinetics, we can determine whether toxicity to or inhibition of the culture had occurred. It can be deduced from eq S11 that the time to reach the maximal resorufin concentration, tbest‑B, is a function of the metabolite/biomass concentration only and is not related to the original concentration of resazurin. According to eq S12, the maximal resorufin concentration, [B]max, is a function of the original concentration of resazurin, [A]0, which should be the actual resazurin concentration in the cell, and not the resazurin concentration initially added in solution. The whole reduction process was suggested to include attachment of resazurin to the
resazurin (A) + M1 k1
→ resorufin (B) + M 2 k2
→ dihydroresorufin (C)
where k1 and k2 are the reaction rate constants for resazurin to resorufin and for resorufin to dihydroresorufin, respectively. The whole reaction process can be described by eq S10, [B] = [A]0[k1/(k2 − k1)](e−k1[M]t − e−k2[M]t). Details of the derivation of this model can be found in the Supporting Information. The optimal time for maximizing resorufin, [B]max = [A]0(k1/ k2)k2/(k2−k1) (eq S12), is tbest‑B = [ln(k1/k2)]/[[M](k1 − k2)] (eq S11). Data Analysis. The fluorescent signals (RFU) obtained were converted to molar concentrations of resorufin by the linear calibration curve (R2 = 0.989; P < 0.0001); RFU = 6608.2[resorufin] + 1.76, in the range of 0−0.1 mM. By fitting resorufin concentrations over time to eq S10, we obtained k1 and k2 to show the kinetics of reduction of resazurin to resorufin and of resorufin to dihydroresorufin, respectively. The values of k1 for the effect of seven chlorophenols on the resazurin reduction assay by E. faecalis or anaerobic sludge were then compared using a one-way analysis of variance (ANOVA) with a Tukey test.
■
RESULTS AND DISCUSSION Resorufin Is Irreversibly Reduced by Anaerobic Sludge and E. faecalis under Anaerobic Conditions. Figure 1 shows the result of resorufin reduction by E. faecalis and fresh mixed anaerobic sludge, where resorufin was quickly reduced under anaerobic conditions. After this experiment, both microplates were exposed to air for 24 h, and the concentration of resorufin increased to the original value, suggesting resorufin had been regenerated. Under anaerobic conditions after the re-addition of fresh mixed anaerobic sludge and subcultured E. faecalis, the regenerated resorufin was reduced again, suggesting that the oxidation/regeneration of resorufin can only happen under aerobic conditions, but under anaerobic conditions, resorufin will be continually reduced to dihydroresorufin, which cannot be oxidized back to resorufin. These observations confirm our assumption about negligible oxidation reactions, and that the system simplifies to the reaction scheme resazurin (A) + M1 → (k1) resorufin (B) + M2 C
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 2. Kinetics of resazurin reduction by E. faecalis under anaerobic conditions. Original resazurin concentrations are (a) 0.05, (b) 0.1, (c) 0.2, and (d) 0.4 mM. For each resazurin concentration, three inoculum densities of E. faecalis (OD600 values of 0.1, 0.2, and 0.3) were investigated with and without 10 mg L−1 spiked PCP. The solid lines are model prediction lines from eq S10.
Table 1. Fitting Results for k1 and k2 for Resazurin Reduction by E. faecalis at Different Inoculum Densities, with and without Spiked PCP resazurin (mM)
inoculum density (OD600)
spiked PCP (mg L−1)
0.4
0.3
10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0
0.2 0.1 0.2
0.3 0.2 0.1
0.1
0.3 0.2 0.1
0.05
0.3 0.2 0.1
k1a (min−1)
k2a (min−1)
R2b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.060 ± 0.008 0.068 ± 0.009 0.085 ± 0.003 0.085 ± 0.002 1.84 × 10−18 2.88 × 10−18 0.013 ± 0.001 0.013 ± 0.001 0.034 ± 0.004 0.021 ± 0.003 1.13 × 10−17 1.23 × 10−17 0.005 ± 0.000 0.008 ± 0.001 0.005 ± 0.001 0.004 ± 0.000 0.014 ± 0.017 0.031 ± 0.005 0.040 ± 0.002 0.040 ± 0.003 0.006 ± 0.001 0.004 ± 0.000 0.004 ± 0.001 0.007 ± 0.001
0.979 0.983 0.972 0.976 0.995 0.998 0.982 0.983 0.990 0.988 0.998 0.993 0.956 0.966 0.986 0.983 0.984 0.998 0.735 0.813 0.870 0.877 0.995 0.993
0.122 0.123 0.095 0.099 0.024 0.036 0.584 0.608 0.185 0.253 0.133 0.178 1.092 1.040 0.589 0.668 0.136 0.285 2.878 2.609 2.692 2.631 0.366 0.518
0.011 0.009 0.002 0.002 0.001 0.003 0.023 0.010 0.018 0.025 0.019 0.021 0.025 0.042 0.030 0.026 0.016 0.025 0.079 0.085 0.057 0.064 0.027 0.029
a
The pseudo-first-order rate constants for resazurin to resorufin and for resorufin to dihydroresorufin (k1 and k2, respectively). The mean constant of three replicates is shown with its standard deviation. bThe coefficient of determination by fitting the mean value of three data replicates to eq S10 (n = 30; critical R2 = 0.214; P = 0.01). D
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 3. Kinetics of resazurin reduction by mixed anaerobic sludge under anaerobic conditions. Original resazurin concentrations are (a) 0.05, (b) 0.1, (c) 0.2, and (d) 0.4 mM. For each resazurin concentration, the diluted fresh mixed anaerobic sludge (VSS at 1.16 g L−1) was investigated with and without 10 mg L−1 spiked PCP. The solid lines are model prediction lines from eq S10 with data from the eighth minute of each study.
reducing equivalents.10 Although a more exact determination of the site(s) of resorufin reduction requires additional study, the same site seems unlikely in the resorufin−dihydroresorufin interconversion according to Talbot et al.,19 and it was shown that NADH is capable of reducing resorufin to dihydroresorufin under anaerobic conditions.15 In this study, the different fitted results for k1 and k2 in each experiment suggested that with the same intracellular metabolite as a reductant (M in eq S10) such as NADH, these two conversions might be mediated by different soluble enzyme(s). Considering anaerobic digestion is highly sensitive to toxicants and needs as much warning as possible, k1 was chosen as the “toxicity indicator”. The highest IC50 ever reported for PCP toxicity to anaerobes is 10 mg L−1.20 However, in the study presented here, 10 mg L−1 PCP was found to have no effect on the reduction of 0.05, 0.1, 0.2, and 0.4 mM resazurin by E. faecalis with inoculum densities (OD600) 0.2 and 0.3 (Figure 2) in the 60 min experiment with similar k1 values (Table 1). At higher inoculum densities, although PCP inhibits the activity of some bacterial cells, the living cells in the reaction were still in excess and considerably greater in number than the inhibited cells. According to eq S11, [M] does not change significantly even with PCP inhibition, ostensibly resulting in no effect on the kinetics of resazurin reduction. However, a spike of 10 mg L−1 PCP delayed the reduction of resazurin by E. faecalis at an OD600 of 0.1, suggesting that at this bacterial density the inhibition of cells by PCP changed the [M] in eq S11, maybe by orders of magnitude, significantly affecting the value of tbest‑B. Therefore, for the application of this fluorescence-based assay to realistic conditions, dilution of the cell sample to lower the density of microorganisms will be necessary.
bacteria, diffusion through the cell wall and membrane, and reaction with metabolites in the cells.10 Therefore, [A]0 could be controlled by the adsorption capacity of living cells, and the diffusivity through the cell wall and membrane, and the rate of diffusion should not be different for the same strain of bacteria. Because the adsorption sites on a bacterial cell are limited,17 there should be a critical [A]0 inside the cell even when the resazurin concentration in the bulk is much higher; this process could be described as “adsorption site limited”. In this study, adding 0.1, 0.2, and 0.4 mM resazurin might be enough to reach the critical resazurin concentration in the cell itself, because under these conditions the same [B]max in panels b−d of Figure 2 was obtained. Moreover, higher concentrations of resazurin were found to affect bacterial growth without affecting dye reduction.18 Therefore, for certain biomass (cells), there should be an optimal resazurin concentration to produce the strongest fluorescent signal. On the basis of the results in Figure 2, 0.1 mM resazurin was determined to be the optimal concentration in this optical measurement of toxicants for the E. faecalis inoculum. For experiments depicted in Figure 2, the resorufin concentration increased initially and after reaching a maximum (with OD600 values of 0.2 and 0.3) decreased slowly, suggesting that before resazurin was consumed completely, resorufin was accumulating in the system; hence, resorufin’s rate of production was greater than its rate of consumption. In Table 1, it can be seen that the fitted values for k1 are greater than k2, proving that the conversion of resazurin to resorufin was faster than the conversion of resorufin to dihydroresorufin, reflecting faster metabolic activity.12 Conversion of resazurin to resorufin is thought to be mediated by soluble enzymes such as diaphorases that use NADH or NAD(P)H as their source of E
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Kinetics of Resazurin Reduction by Mixed Anaerobic Sludge: Effect of Original Resazurin Concentration and Spiked PCP. The reduction of 0.05−0.4 mM resazurin in mixed anaerobic sludge is shown in Figure 3. Unlike resazurin reduction with a pure culture of E. faecalis in which the level of resorufin increased exponentially from the beginning of the reaction, a lag phase occurred in the first 8 min before resazurin was reduced by anaerobic sludge. In this case, only data from the eighth minute of each study were fitted, and these results are summarized in Table 2. On the basis of R2, the model (eq
associated sediment−water interactions. Anaerobic sludge is comprised of a microbial consortium and organic and inorganic matter held together in a matrix, e.g., sludge granules and flocs, formed by exocellular biopolymer and cations. Within flocs or granules, diffusion is usually the primary mass transfer mechanism with a diffusive barrier to solute molecules approaching or leaving the biological cell surface23 and thus may strongly affect the overall reaction rate.24 This diffusive barrier for resazurin or PCP to penetrate into the cell wall/ membrane could explain the lag phase for resorufin obtained with anaerobic sludge. The rate of resazurin conversion depends not only on the metabolic status of a cell but also on the permeability of the cell envelope, including the diffusive barrier. However, no matter how significant the diffusional barrier is, the resazurin reduction assay shows considerable potential in identifying toxicants to anaerobic digestion by its change in reduction kinetics. Identifying the Toxicity Difference among Seven Chlorophenols to a Single Anaerobic Strain and Mixed Anaerobic Sludge. Figure 4 shows the results of resazurin reduction in OD600 = 0.1 E. faecalis (a) and 1:1 (v:v) diluted fresh anaerobic sludge (b) spiked with seven types of chlorophenols compared to a control, and the k1 values of the model (eq S10) are summarized in Table 3. A one-way ANOVA (including a Tukey test) was used to evaluate whether
Table 2. Fitting Results for k1 and k2 for Resazurin Reduction by Diluted Fresh Mixed Anaerobic Sludge (VSS at 1.16 g L−1) with and without Spiked PCP [resazurin] (mM)
spiked PCP (mg L−1)
0.4
10 0 10 0 10 0 10 0
0.2 0.1 0.05
k1a (min−1) 0.180 0.195 0.207 0.237 0.282 0.372 0.423 0.625
± ± ± ± ± ± ± ±
0.019 0.021 0.035 0.047 0.044 0.039 0.026 0.033
k2a (min−1)
R2b
± ± ± ± ± ± ± ±
0.966 0.964 0.997 0.931 0.956 0.933 0.991 0.820
0.070 0.082 0.044 0.119 0.022 0.170 0.013 0.213
0.004 0.004 0.003 0.011 0.001 0.025 0.001 0.033
a
The pseudo-first-order rate constants for resazurin to resorufin and for resorufin to dihydroresorufin (k1 and k2, respectively). The mean constant of three replicates is shown with its standard deviation. bThe coefficient of determination by fitting the mean value of three data replicates to eq S10 (n = 30; critical R2 = 0.214; P = 0.01).
S10) can also well describe the kinetics of resazurin reduction in mixed anaerobic sludge by ignoring the lag phase. Without addition of PCP, the concentration of resorufin in the control samples increased initially and after reaching a maximum decreased slowly, and for 0.05 and 0.1 mM resazurin addition down to almost background level as it was completely reduced to dihydroresorufin. On the basis of the kinetics of resazurin reduction summarized in Table 2, except at 0.4 mM resazurin, 10 mg L−1 PCP significantly inhibited sludge activity compared to the control by reducing both the kinetics of reduction of resazurin to resorufin (k 1 ) and that of resorufin to dihydroresorufin (k2). The sludge used in this part of the experiment was collected 1 h after the seed reactor was fed, and this fresh sludge showed high activity in reducing resazurin to resorufin, and then to dihydroresorufin, with fitted values of k1 larger than the values of k2 (Table 2) as obtained previously with pure E. faecalis. Therefore, k1 is also a good “indicator” for the effect of toxicants on anaerobic digestion. On the basis of the results in Figure 3, and considering maximal resorufin production and time to reach its maximal value, 0.1 mM resazurin was also determined to be the optimal concentration for the optical measurement with mixed anaerobic sludge. Some authors have suggested that the reduction of resazurin to resorufin might occur in the mitochondria or ribosomes, on the surface of the plasma membrane, or maybe even in the culture medium itself,21 while living cells but not medium were found to be essential for reducing resazurin to highly fluorescent resorufin.10 In a more complex environment, Haggerty et al.22 reported that resazurin is irreversibly and rapidly reduced to resorufin in colonized sediment also with pseudo-first-order behavior, but both resazurin and resorufin are significantly affected by sorption, indicating that the resazurin assay is sensitive to microbiological activity and
Figure 4. Application of the resazurin reduction assay to identify the toxicity difference among seven chlorophenols. The seven chlorophenols are 2-monochlorophenol (2-CP), 3-monochlorophenol (3CP), 4-monochlorophenol (4-CP), 2,3-dichlorophenol (2,3-DCP), 2,5-dichlorophenol (2,5-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP). Their toxicity difference was studied with (a) the pure facultative anaerobic strain E. faecalis at an OD600 of 0.1 and (b) diluted fresh mixed anaerobic sludge with VSS at 1.16 g L−1. F
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Table 3. k1 (reaction rate constants for resazurin to resorufin) in Seven Chlorophenol Spiked Systems with a Control and P Values of the Statistical Analysis Comparing k1 Values among Seven Chlorophenols with a Control (Tukey test included in the one-way ANOVA; shaded area, in E. faecalis; unshaded area, in anaerobic sludge)
a Same description as in Table 1. *The significance of the bold numbers indicates the current result is NOT significantly different by comparing the column to the row according to the Tukey test at P < 0.05.
analysis of k1 values summarized in Table 3, and this order is similar to that of the octanol/water (logP) partition coefficient.1 Nevertheless, for chlorophenols with the same number of chlorine groups, it has been suggested that the position of the substitution influences toxicity, and the sequence in decreasing order of toxicity was meta > para ≫ ortho,25 although this was not observed in the study presented here. Quantification of the biotoxicity of seven chlorophenols on anaerobic sludge in just 30 min using the optical assay described here indicates its considerable potential for use in the real-time monitoring of toxicants for anaerobic digestion. The toxic action of chlorophenols on a living cell was proposed to disrupt the proton gradient through the membrane, and interfering with cellular energy transduction, thereby decreasing the rate of cell growth due to an uncoupling of the catabolic and anabolic reactions,20 and accordingly whittling down the metabolites that will be involved in resazurin reduction. Heavy metals are the most frequently found toxicants for anaerobic digestion, and it is believed that heavy metals manifest their toxicity through their disruption of enzyme function and structure by binding with thiol and other groups on protein molecules, or by replacing natural metals in enzyme prosthetic groups in cells.26 Therefore, on the basis of the toxicity mechanisms of heavy metals and chlorophenols, with the kinetics of resazurin reduction, the biotoxicity of heavy metal ions can also be evaluated using this bioassay. Considering their toxicity mechanisms for anaerobic digestion,1 many other toxicants, such as halogenated aliphatics and ammonia, can be studied by the resazurin reduction bioassay. However, we do not suggest applying this bioassay to the toxicity of nanomaterials because there have already been
the spiked toxicant had a significant effect on the kinetics of reduction of resazurin to resorufin to assess the toxicity difference (if any) among seven chlorophenols to a single anaerobic strain and fresh mixed anaerobic sludge. The one-way ANOVA test shows that spiking all the chlorophenols had a significant effect on the kinetics of resazurin reduction, and the difference among the seven chlorophenols based on the Tukey test is summarized in Table 3. From Figure 4a, there appears to be no obvious difference among the seven chlorophenols compared to the control system. However, by statistically analyzing the model fitting results, we can tell with resazurin reduction by E. faecalis (shaded area in Table 3) that there are no significant differences by spiking 2-CP, 3-CP, and 4-CP with P values of 0.904, 0.976, and 0.534, respectively, which are larger than 0.05, the statistically significant threshold. The same conclusion can be drawn among 2,3-DCP, 2,5-DCP, and 2,4,6TCP with resazurin reduction by E. faecalis. However, in the fresh mixed anaerobic sludge system (clear area in Table 3), the seven chlorophenols show significant differences, except between PCP and 2,4,6-TCP, 2-CP and the control, 3-CP and 4-CP, and 2-CP and 3-CP, which have P values of 0.998, 0.823, 0.823, and 0.141, respectively. These results suggest that all seven chlorophenols show different toxicities to mixed anaerobic sludge except for the pair with a P value of >0.05 (Table 3). In this study, all the chlorophenols showed a significant effect on resazurin reduction with both the pure strain and the sludge, compared to the control, except for 2-CP in the anaerobic sludge [P = 0.823 (Table 3)]. The order of toxicity of the chlorophenols in anaerobic sludge was PCP ≈ 2,4,6-TCP > 2,5DCP > 2,3-DCP > 4-CP ≈ 3-CP ≈ 2-CP based on a statistical G
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
(5) Rozzi, A.; Tomei, M. C.; Di Pinto, A. C.; Limoni, N. Monitoring toxicity in anaerobic digesters by the Rantox biosensor: theoretical background. Biotechnol. Bioeng. 1997, 55 (1), 33−40. (6) Pollice, A.; Rozzi, A.; Concetta Tomei, M. C.; Di Pinto, A. C.; Laera, G. Inhibiting effects of chloroform on anaerobic microbial consortia as monitored by the Rantox biosensor. Water Res. 2001, 35 (5), 1179−1190. (7) Kaur, A.; Kim, J. R.; Michie, I.; Dinsdale, R. M.; Guwy, A. J.; Premier, G. C. Sustainable Environm Res, C., Microbial fuel cell type biosensor for specific volatile fatty acids using acclimated bacterial communities. Biosens. Bioelectron. 2013, 47, 50−55. (8) Liu, Z.; Liu, J.; Zhang, S.; Xing, X.-H.; Su, Z. Microbial fuel cell based biosensor for in situ monitoring of anaerobic digestion process. Bioresour. Technol. 2011, 102 (22), 10221−10229. (9) Pratten, M.; Ahir, B. K.; Smith-Hurst, H.; Memon, S.; Mutch, P.; Cumberland, P. Primary cell and micromass culture in assessing developmental toxicity. Methods Mol. Biol. (N. Y., NY, U. S.) 2012, 889, 115−146. (10) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 2000, 267 (17), 5421− 5426. (11) Wirth, R.; Kovacs, E.; Maroti, G.; Bagi, Z.; Rakhely, G.; Kovacs, K. L. Characterization of a biogas-producing microbial community by short-read next generation DNA sequencing. Biotechnol. Biofuels 2012, 5, 41. (12) Chen, J. L.; Ortiz, R.; Xiao, Y.; Steele, T. W. J.; Stuckey, D. C. Rapid fluorescence-based measurement of toxicity in anaerobic digestion. Water Res. 2015, 75, 123−130. (13) Nakayama, G. R.; Caton, M. C.; Nova, M. P.; Parandoosh, Z. Assessment of the alamar blue assay for cellular growth and viability in vitro. J. Immunol. Methods 1997, 204 (2), 205−208. (14) Chen, P.; Zhou, X.; Shen, H.; Andoy, N. M.; Choudhary, E.; Han, K. S.; Liu, G.; Meng, W. Single-molecule fluorescence imaging of nanocatalytic processes. Chem. Soc. Rev. 2010, 39 (12), 4560−4570. (15) Zhao, B.; Ranguelova, K.; Jiang, J.; Mason, R. P. Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red. Free Radical Biol. Med. 2011, 51 (1), 153−159. (16) Natto, M. J.; Savioli, F.; Quashie, N. B.; Dardonville, C.; Rodenko, B.; de Koning, H. P. Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis. J. Antimicrob. Chemother. 2012, 67 (4), 933−943. (17) Muyima, N. O.; Momba, M.; Cloete, T. Biological methods for the treatment of wastewaters. Microbial community analysis: The key to the design of biological wastewater treatment systems; Cloete, T. E., Muyima, N. O., Eds.; IAWQ Scientific and Technical Report; 1997, Vol. 5, pp 1−24. (18) Elavarasan, T.; Chhina, S. K.; Parameswaran (Ash), M.; Sankaran, K. Resazurin reduction based colorimetric antibiogram in microfluidic plastic chip. Sens. Actuators, B 2013, 176, 174−180. (19) Talbot, J. D.; Barrett, J. N.; Barrett, E. F.; David, G. Rapid, stimulation-induced reduction of C12-resorufin in motor nerve terminals: Linkage to mitochondrial metabolism. J. Neurochem. 2008, 105 (3), 807−819. (20) Chen, Y.; Cheng, J. J.; Creamer, K. S. Inhibition of anaerobic digestion process: A review. Bioresour. Technol. 2008, 99 (10), 4044− 4064. (21) Quent, V. M. C.; Loessner, D.; Friis, T.; Reichert, J. C.; Hutmacher, D. W. Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. J. Cell. Mol. Med. 2010, 14 (4), 1003−1013. (22) Haggerty, R.; Argerich, A.; Martí, E. Development of a “smart” tracer for the assessment of microbiological activity and sedimentwater interaction in natural waters: The resazurin-resorufin system. Water Resour. Res. 2008, 44 (4), W00D01. (23) Kapellos, G. E.; Alexiou, T. S.; Payatakes, A. C. A multiscale theoretical model for diffusive mass transfer in cellular biological media. Math. Biosci. 2007, 210 (1), 177−237.
reports about the reaction between nanoparticles and resazurin,27 and the interference of carbon nanotubes in the resazurin reduction assay through quenching of fluorescence.28 In summary, this study provides the first comprehensive insight into the kinetics of resazurin reduced to resorufin, and eventually to dihydroresorufin under anaerobic conditions. Considering the simulation results of resazurin reduction by a pure facultative culture, E. faecalis, and a mixed anaerobic sludge, with and without spiked PCP, the kinetic model developed can describe these reactions well. Hence, the pseudo-first-order rate constant for resazurin to resorufin, k1, can be used as a “toxicity indicator” to identify toxicants to anaerobic digestion. Using this rate constant, we identified toxicity differences among seven chlorophenols, which compared well with the reported toxicity sequences of various chlorophenols in the literature. Besides the potential for monitoring and identifying toxicants for anaerobic digestion, compared to the previously developed bioassays, the use of a fluorescent microplate reader in this bioassay affords the opportunity to collect fine resolution data with respect to time and to process many samples and/or replicates. Taken together, these findings show that this resazurin-based fluorescence measurement not only can detect toxicants but also can determine the toxicity differences among a variety of similar toxicants.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b03050. Details of the derivation of the resazurin reduction kinetic model (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*Phone: +44 207 5945591. Fax: +44 15 317453. E-mail: d.
[email protected]. *Phone: +65-6592-7594. Fax: +65-6790-9081. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This research was supported by the Singapore National Research Foundation under its Environmental & Water Technologies Strategic Research Programme and administered by the Environment & Water Industry Programme Office (EWI) of the PUB.
■
REFERENCES
(1) Chen, J. L.; Ortiz, R.; Steele, T. W. J.; Stuckey, D. C. Toxicants inhibiting anaerobic digestion: A review. Biotechnol. Adv. 2014, 32 (8), 1523−1534. (2) Owen, W. F.; Stuckey, D. C.; Healy, J. B., Jr; Young, L. Y.; McCarty, P. L. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res. 1979, 13 (6), 485−492. (3) Rozzi, A.; Castellazzi, L.; Speece, R. E. Acetoclastic methanogenic activity measurement by a titration bioassay. Biotechnol. Bioeng. 2002, 77 (1), 20−26. (4) Ma, X. Y.; Wang, X. C.; Ngo, H. H.; Guo, W.; Wu, M. N.; Wang, N. Bioassay based luminescent bacteria: Interferences, improvements, and applications. Sci. Total Environ. 2014, 468−469, 1−11. H
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology (24) Novak, J. T.; Sadler, M. E.; Murthy, S. N. Mechanisms of floc destruction during anaerobic and aerobic digestion and the effect on conditioning and dewatering of biosolids. Water Res. 2003, 37 (13), 3136−3144. (25) Pera-Titus, M.; García-Molina, V.; Baños, M. A.; Giménez, J.; Esplugas, S. Degradation of chlorophenols by means of advanced oxidation processes: a general review. Appl. Catal., B 2004, 47 (4), 219−256. (26) Soldatkin, O. O.; Kucherenko, I. S.; Pyeshkova, V. M.; Kukla, A. L.; Jaffrezic-Renault, N.; El’skaya, A. V.; Dzyadevych, S. V.; Soldatkin, A. P. Novel conductometric biosensor based on three-enzyme system for selective determination of heavy metal ions. Bioelectrochemistry 2012, 83, 25−30. (27) Xu, W.; Kong, J. S.; Yeh, Y.-T. E.; Chen, P. Single-molecule nanocatalysis reveals heterogeneous reaction pathways and catalytic dynamics. Nat. Mater. 2008, 7 (12), 992−996. (28) Breznan, D.; Das, D.; MacKinnon-Roy, C.; Simard, B.; Kumarathasan, P.; Vincent, R. Non-specific interaction of carbon nanotubes with the resazurin assay reagent: Impact on in vitro assessment of nanoparticle cytotoxicity. Toxicol. In Vitro 2015, 29 (1), 142−147. (29) Twigg, R. S. Oxidation-reduction aspects of Resazurin. Nature 1945, 155 (3935), 401−402.
I
DOI: 10.1021/acs.est.5b03050 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
