Effect of Nickel and Cadmium Speciation on Nitrification Inhibition

Picker Engineering Program, Smith College,. Northampton, Massachusetts 01063. Heavy metals have been postulated to cause significant nitrification inh...
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Environ. Sci. Technol. 2002, 36, 3074-3078

Effect of Nickel and Cadmium Speciation on Nitrification Inhibition Z H I Q I A N G H U , † K A R T I K C H A N D R A N , †,‡ DOMENICO GRASSO,§ AND B A R T H F . S M E T S * ,† Environmental Engineering Program, University of Connecticut, Storrs, Connecticut 06269-2037, and Picker Engineering Program, Smith College, Northampton, Massachusetts 01063

Heavy metals have been postulated to cause significant nitrification inhibition. Using biomass from a well-controlled continuously operated lab-scale nitrifying bioreactor, the effect of nickel and cadmium on ammonium and nitrite oxidation was quantified. The extent of inhibition was calculated from the kinetics of ammonium oxidation and nitrite oxidation, inferred from maximum specific oxygen uptake rates (SOUR) measured in batch respirometric assays. Nickel and cadmium inhibited ammonium oxidation but not nitrite oxidation up to total analytical concentrations of approximately 1.0 mM. Metal fractions (total and free ion) were correlated with the extent of ammonium oxidation inhibition in the presence of various metal complexing agents (EDTA, NTA, citrate, SO42-). Inhibition was not a function of the total analytical metal concentration but strongly correlated with free cation concentration, [Ni2+] or [Cd2+]. This relationship could be described by either an empirical noncompetitive inhibition model for [Ni2+] or a linear model in the case of [Cd2+]. Furthermore, the free Ni2+ or Cd2+ concentrations could be modulated by the addition of a strong chelating agent (e.g., EDTA), resulting in reduced deleterious effects. However, at elevated doses, EDTA itself impaired nitrification. In sum, predictions and mandatory strategies of nitrification inhibition by heavy metals should be based on free cation concentrations and not on total metal concentrations.

Introduction Wastewater treatment plants typically rely on the autotrophic nitrification/heterotrophic denitrification sequence to remove inorganic nitrogen. Because of the low growth rate of the nitrifying microorganisms and their sensitivity to a number of environmental conditions such as pH, dissolved oxygen concentration, and temperature (2-5), nitrification [involving the conversion of ammonium-nitrogen (NH4+-N) to nitrite (NO2- -N) and nitrate (NO3- -N)] is generally considered the controlling step in biological nitrogen removal (1). A variety of organic and inorganic chemical species can also adversely affect the specific growth rate of the nitrifying bacteria (6, 7). Heavy metals, often associated with industrial * Corresponding author phone: (860)486 2270; fax: (860)486-2298; e-mail: [email protected]. † University of Connecticut. ‡ Present address: Metcalf & Eddy, 60 East 42 St., New York, NY 10165. § Smith College. 3074

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wastewater contributions, have been postulated as a cause of nitrification inhibition (8, 9). Unlike organic chemicals, heavy metals are not biodegradable and can bioaccumulate to potentially inhibitory concentrations (10). The exact mechanism by which heavy metals interfere with nitrifying activity has not yet been elucidated. In general, however, it is believed that the first step in the microbial response to toxic heavy metals is the uptake of free metal cations via a nonspecific metal inorganic transport system (11). Once inside the cell, heavy metals can interact with thiol groups and destroy protein structure and function. In addition, heavy metals may interfere with physiologically important ions (e.g., Cd2+ with Zn2+ or Ca2+; Ni2+ with Fe2+; Zn2+ with Mg2+) and inhibit the function of the respective physiological cations (11). The importance of metal speciation in toxicity has been widely recognized (12-14). If extracellular mass transfer (i.e., physical transport) and coordination reactions are rapid as compared with biological uptake processes, a pseudoequilibrium can be established between the metal in the bulk solution and on the microbial surface (15). In such cases, heavy metal toxicity is directly proportional to the free ion activity in solution, which is the basis of the free ion activity model (FIAM) (16). The activity of the free metal cation can, however, be significantly altered via complexation with aqueous ligands (17, 18) or adsorption of metals directly to biomass and their extracellular biopolymers (19-21). The literature, to date, is composed of primarily qualitative evidence indicating that the “free” metal cation concentration is a good predictor of biological response (20, 22). Moreover, the adequacy of the FIAM has rarely been demonstrated in relation to bacterial activity (12, 14). The objectives of this research were therefore to quantitatively determine the effect of two representative heavy metals, nickel and cadmium, on nitrification activity and to better understand the relative importance of total and free metal cation concentrations with regard to nitrification inhibition.

Experimental Section Nitrifying Bioreactor. Nitrifying biomass was cultivated in a continuously stirred tank reactor (10 L) operated at solids retention time (SRT) of 20 d and hydraulic retention time (HRT) of 1 d. The reactor was fed an inorganic medium devoid of organic carbon, with ammonium (300 mg/L N, (NH4)2SO4) as the sole energy source with requisite macro- and micronutrients (Table 1). Sodium carbonate (1 M) was intermittently added to maintain the reactor pH at 7.4 ( 0.1 and fulfilled both carbon and alkalinity requirements. Filtered laboratory air was provided to ensure adequate mixing and aeration. Reactor performance was monitored via reactor and effluent COD, effluent NH4+-N, NO2- -N, and NO3- -N concentrations. Upon attainment of steady state [more than 60 d (3 SRTs) after reactor start up with the effluent concentrations of NH4+-N and NO2- -N nondetectable and NO3- -N approximately 300 mg/L with a coefficient of variation less than 10%], mixed liquor was periodically withdrawn from reactor and used for batch studies. Nitrifier Substrate Oxidation Activity. Maximum specific substrate oxidation rates were measured in duplicate using batch extant respirometric assays described elsewhere (23). Briefly, biomass aliquots (50 mL) were collected from the continuous reactor. The assay was performed at a final pH of 7.0 ( 0.05 in replicate 50-mL water-jacketed glass vessels, maintained at 25 ( 0.5 °C. Biomass suspensions were aerated with pure oxygen gas before substrate aliquots (NH4+-N or NO2- -N) were injected. Initial concentrations of 5 mg/L 10.1021/es015784a CCC: $22.00

 2002 American Chemical Society Published on Web 05/30/2002

TABLE 1. Composition of Reactor Influent concentrations in reactor influent compound MgSO4‚7H2O CaCl2‚2H2O NaCl (NH4)2SO4 K2HPO4 FeSO4‚7H2O MnSO4‚H2O (NH4)6Mo7O24‚4H2O CuCl2‚2H2O ZnSO4‚7H2O NiSO4‚6H2O

mg/L 280 120 600 1500 27.2 3.3 3.3 0.8 0.8 1.7 0.3

a Total NH + ) 22.76 mM. 4 11.99 mM.

b

cations (mM) Mg2+

1.14 0.82 Ca2+ 10.34 Na+ 22.72 NH4+ a 0.31 K+ 0.01 Fe2+ 0.02 Mn2+ 0.04 NH4+ a 0.005 Cu2+ 0.006 Zn2+ 0.001 Ni 2+

anions (mM) 1.14 SO42- b 1.64 Cl- c 10.34 Cl- c 11.36 SO42- b 0.16 HPO420.01 SO42- b 0.02 SO42- b 0.001Mo7O2460.01 Cl- c 0.006 SO42- b 0.001 SO42- b

Total SO42- ) 12.54 mM. c Total Cl- )

NH4+-N and 10 mg/L NO2- -N were determined to yield substrate-independent maximal oxygen uptake rates (data not shown). A decrease in the dissolved oxygen (DO) level in the vessel due to substrate oxidation was measured by a DO probe (YSI model 5331, Yellow Springs, OH) and continuously recorded at 4 Hz by a personal computer interfaced to a DO monitor (YSI model 5300, Yellow Springs, OH). To maintain pH during ammonium oxidation, buffering capacity was provided by adding 20 mM MOPS [3-(Nmorpholino)propanesulfonic acid, pH adjusted to 7] to the cultures. We concluded that MOPS had insignificant complexation potential with cadmium, experimentally inferred from the fact that constructed standard curves of free cadmium cation concentrations in the range of 10-5-10-3 M with and without 20 mM MOPS applied were identical (data not shown, t-test, p ) 0.03). Furthermore, MOPS had no inhibitory effect on nitrifying activity based on a comparison of ammonium oxidation kinetics inferred from assays in a 20 mM MOPS buffer versus a 4 mM phosphate buffer (23) (data not shown, t-test, p ) 0.05). The effect of nickel and cadmium on nitrification kinetics was investigated in the absence or presence of specifically added metal complexing agents. The complexing agents sulfate, citrate, nitrilotriacetate (NTA), and ethylenediaminetetraacetate (EDTA) were chosen to cover a range of Ni2+ and Cd2+ complex formation constants from 102.3 (NiSO4) to 1020.4 (NiEDTA2-). While a ligand like SO42- has affinity for metals, it is unlikely that metal-sulfate complexes are formed through multidentate coordination. Hence, strictly speaking, sulfate is not a chelating agent. For the purposes of this paper, we refer to all tested ligands as complexing agents. Complexing agents were mixed with Ni or Cd stock solutions to yield final concentrations of complexing agents, Ni, and Cd in the range of 0-10, 0-1, and 0-0.8 mM, respectively. The concentration of the free metal cation, Ni2+or Cd2+, could thus be manipulated at constant analytical Ni or Cd concentrations by varying the type and concentration of complexing agent or at constant concentration of complexing agents by varying the analytical Ni or Cd concentrations. All batch inhibition assays were conducted with a consistent metal exposure time. That is, after the stock solutions containing Ni or Cd in the absence or presence of complexing agents were spiked into batch vessels a standard substrate oxidation assay ensued. The actual time elapsed between metal spike and ammonium spike was approximately 15 min. Calculation of the Extent of Nitrification Inhibition. Although the term inhibition subsumes a reduction in some specific activity measure and toxicity commonly refers to complete inactivation, the literature has not consistently distinguished between inhibition and toxicity (24). For the

purposes of this paper, we will follow the common interpretation of inhibition to describe the effect of heavy metal on nitrification. Inhibition of biological activity was inferred from the difference between the measured maximum specific oxygen uptake rate (SOUR) in the absence (SOURcontrol) and presence (SOURsample) of the test heavy metal:

% inhibition ) (SOURcontrol - SOURsample)/SOURcontrol × 100% (1) Calculation of Metal Speciation. Ni and Cd speciation was determined with the MINEQL+ (version 4.5) chemical equilibrium speciation algorithm (25), with incorporation of conditional stability constants for the metal biomass species. Correction of stability constants for ionic strength was made using the Davies equation at 25 °C (25). Total inorganic carbon (TIC) concentration in the continuous reactor and respirometric vessels was measured at 1.75 ( 0.27 and 1.54 ( 0.21 mM, respectively. On the basis of kinetic experiments, otavite (CdCO3(s)) formation was not expected in the time frames of the inhibition studies conducted here. After adding 1.5 mM carbonate and 20 mM MPOS at pH 7 to a 0.7 mM CdCl2 solution and allowing the reaction to proceed for 2 weeks, a decrease of only 3.5% in Cd2+ concentration was recorded. Hence, CdCO3(s) as potential species was removed from the MINEQL+ database to represent our systems. Conditional stability constants for Cd biomass were estimated from metal partitioning experiments with biomass at 764 ( 5 mg/L COD and CT,Cd at 0, 20, 40, 60, 80, and 100 mg/L. From these experiments, a linear partitioning between soluble and cell-associated metal was inferred (CCd,soluble ) 0.84 ( 0.01CT,Cd, R2 ) 1.0). Furthermore, a linear relationship between soluble and cationic metal was measured over the same experimental range (CCd2+ ) 0.51 ( 0.02 CT,Cd, R2 ) 0.98). The stability constant was subsequently calculated as (CT,Cd - CCd,soluble)/(CCd2+)(Cbiomass) with all concentrations expressed in molar units (with an empirical formula of biomass considered as C5H7O2N (24), 1 mol of biomass COD ) 32 g). Similar procedures were applied to estimated Ni biomass partitioning. Resulting biomass stability constants were 101.1 and 101.4 for Cd and Ni, respectively. Complexation of Cd2+ with MOPS was insignificant as mentioned above and could be omitted from equilibrium calculations. Similarly, Ni-MOPS complexation was not considered because Ni and Cd have similar formation constants for a range of ligands (26). Complexation of metals with soluble organic chelating agents present in the reactor medium was considered insignificant because the feed medium was devoid of organic compounds and the soluble organic compound concentration in the reactor was below 20 mg/L as COD constituting less than 2% of reactor total COD concentration. All other formation constants were directly available in the MINEQL+ database. Analytical Procedures. Ni and Cd concentrations were measured according to standard methods (27) by flame atomic absorption spectrometry (model 5100, Perkin-Elmer Co.) with method detection limits of 1.7 and 0.1 µM for Ni and Cd, respectively. Free cadmium cation concentration was measured by an ion-selective electrode (ISE, Orion model 94-48, MA) with a method detection limit of 5 µM. The Cd ISE was weekly calibrated with CdNO3 standards (0.01, 0.1, and 1 mM) prepared with pH 4.0 DI water yielding log-linear calibration curves. Biomass concentrations were measured as chemical oxygen demand using commercially available reagents (HACH COD vials, 0-1500 mg/L). Total inorganic carbon was measured by total organic carbon analyzer (model TOC-5000A, Shimadzu). VOL. 36, NO. 14, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Effect of nickel and cadmium on ammonium and nitrite oxidation kinetics as measured by specific oxygen uptake rates with ammonium or nitrite as substrate. Circles: Ni; squares: Cd. NH4+-N oxidation (b, 9); NO2- -N oxidation (O, 0). Error bars indicate one standard deviation.

FIGURE 3. Inhibition of NH4+-N oxidation as a function of total nickel concentration CT,Ni or calculated nickel cation concentration [Ni2+] in the presence of complexing agents, EDTA, NTA, citrate, or sulfate. Filled symbols (b, 9) refer to experiments where total metal concentration CT,M was kept constant while varying [ligand]. Open symbols (O, 0) refer to experiments where [ligand] was kept constant while varying CT,M. Metal concentrations are expressed as total concentrations (b, O), or as free metal cation concentration (9, 0). Dashed and continuous lines are best-fit linear regressions with respect to total metal and free metal cation concentrations, respectively.

FIGURE 2. Comparison of predicted (MINEQL+) and analytical (ionselective electrode) cadmium cation concentrations. The line of equality is indicated. Precautions were taken to avoid trace metal contamination. Glassware and plastic ware, when appropriate, were soaked in 1 M HNO3 overnight and rinsed with 5 vol of Milli-Q purified water before use.

Results Ammonium and Nitrite Oxidation Kinetics. The specific ammonium oxidation rate (SOURNH4) decreased as the applied nickel dose to the nitrifying biomass increased (Figure 1). In contrast, the specific nitrite oxidation rate (SOURNO2) was observed to be less sensitive to Ni concentrations tested (up to 1.7 mM). A similar inhibition pattern was observed for Cd (Figure 1). At a dose of 1 mM each, Ni and Cd inhibited ammonium oxidation approximately 30% and 70%, respectively. The higher sensitivity of ammonium oxidation versus nitrite oxidation is consistent with earlier observations of nitrification inhibition (7, 28). Accuracy of MINEQL+ Model Predictions. The MINEQL+ algorithm provided excellent predictions of the analytical free Cd2+ concentration (Figure 2). In the absence of an ionselective Ni electrode, such analysis was not possible for Ni, but MINEQL+ predictions were considered valid based on the verification with Cd. Inhibitory Effect Related to Total Nickel or Cadmium. In the presence of complexing agents, very poor correlations were observed between the total Ni concentration and the inhibitory effect on ammonium oxidation (Figure 3). Similarly, poor correlations (except in the presence of sulfate) were observed between inhibition and total Cd concentration (Figure 4). Hence, the total analytical aqueous Ni or Cd concentrations were not good predictors of the metals’ effect on nitrification kinetics. These results agree with reports 3076

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FIGURE 4. Inhibition of NH4+-N oxidation as a function of total cadmium concentration CT,Cd or measured cadmium cation concentration [Cd2+] in the presence of complexing agents, EDTA, NTA, citrate, or sulfate. Symbols are as defined in Figure 3. Error bars on [Cd2+] measurement indicate one standard deviation. indicating that the total aqueous metal concentrations do not predict their toxic effects (18, 22). Inhibitory Effect Related to Free Nickel or Cadmium. Free metal cations are generally thought to be the most toxic metal species (13, 22). According to the free ion activity model (16), the extent of inhibition is correlated to the concentration of the free metal cation irrespective of the total aqueous metal concentrations. This prediction was supported by our results. The extent of inhibition of NH4+-N oxidation kinetics correlated closely with the free Ni cation concentration (Figure 3) or free Cd cation concentration (Figure 4),

FIGURE 5. Inhibition of NH4+-N oxidation as a function of (a) calculated free nickel cation concentration, [Ni2+], or (b) measured free cadmium cation concentration, [Cd2+], showing all experimental data and best-fit with (a) noncompetitive inhibition model (% inhibition ) 100/(1 + Ki/I)) or (b) linear inhibition model (% inhibition ) 188I). Symbols are as defined in Figures 3 and 4. Ki and I are the half-inhibition coefficient and free metal cation concentration, respectively. indicating that free metal cation was most likely responsible for the observed inhibitory effects on nitrifying activity. While an empirical noncompetitive inhibition model (shown in Figure 5a) provided a good fit (R2 ) 0.87 vs R2 ) 0.76 for linear model) to the full collection of Ni inhibition experiments, a simple linear model adequately fit the full collection of Cd inhibition experiments (R2 ) 0.94 vs R2 ) 0.89 for noncompetitive inhibition model) (Figure 5b) when the free metal cation was considered as inhibitor. The EC50, defined as the concentration of free metal cation causing 50% inhibition, was estimated at 1.00 and 0.27 mM for Ni and Cd, respectively. Reduction of Inhibition by Complexing Agents. Addition of complexing agents reduced the biological effect of Ni, but the actual trend was agent-specific (Figure 6a). For nickel, MINEQL+ calculations indicated that the 1:1 Ni complexes (e.g., NiEDTA2-, NiNTA-, NiCitrate- and NiSO4) were the predominant complexed species. At 1 mM concentration, EDTA completely eliminated nickel’s biological effect, and no inhibition was observed. Hence, our experimental findings are consistent with other observations that NiEDTA2- is essentially inert and nonbioavailable (30). Although NTA forms weaker complexes with Ni than EDTA [formation constants of Ni-EDTA and Ni-NTA are 1020.4 and 1012.8, respectively (26)], NTA addition had a similar quantitative effect on relieving Ni inhibition. MINEQL+ calculations indicated, however, that Ni2+ concentrations were similar in both cases, probably because EDTA also formed strong complexes with abundant Ca and Mg cations in solution. Addition of 1 mM citrate only moderately reduced nickel’s biological effect (12% inhibition of NH4+-N oxidation). At 2 mM citrate concentration, inhibition by Ni was completely obviated. Addition of 1 mM sulfate did not impact nickel’s biological effect. Even at 10 mM sulfate, inhibition persisted. As NiSO4 is characterized

FIGURE 6. Inhibition of NH4+-N oxidation as a function of complexing agents EDTA, NTA, citrate, or sulfate in the presence of (a) 1 mM Ni or (b) 0.7 mM Cd. Horizontal lines represent average percent of inhibition in the presence of (a) 1 mM Ni or (b) 0.7 mM Cd in the absence of complexing agents. by a weak thermodynamic equilibrium (K ) 102.3), high concentrations of NiSO4 species continue to result in high concentration of the free Ni cation, the probable species responsible for nitrification inhibition. Similar to the results observed in nickel experiments, addition of complexing agents reduced the biological effect of Cd, and the extent of reduction was governed by the concentration and the affinity of ligands for the free Cd cation (Figure 6b). For example, citrate forms weaker complexes with Cd than with Ni [formation constants of CdCitrate- and NiCitrate- are 104.6 (25, 29) and 106.6 (25), respectively]. As a result, even at 8 mM citrate concentration, inhibition by Cd was only partially relieved. Although addition of complexing agents reduced the biological effect of Ni or Cd, significant inhibition was observed at increased doses of EDTA (Figure 6). Clearly, a narrow range in EDTA doses existed to completely relieve nitrification inhibition.

Discussion To date, notwithstanding substantial qualitative evidence, the FIAM has not been quantitatively confirmed and has rarely been evaluated in relation to bacterial activity. Our results have quantitatively shown that there is a correlation between the biological response (e.g., nitrification inhibition) of nickel or cadmium for a microorganism and the thermodynamic activity of the free metal cation in solution. Thus, free nickel or free cadmium cations might be used to infer nitrification inhibition in biological reactors. However, in certain cases the free ion activity paradigm does not appear valid (22, 31). For example, metal complexes with selected hydrophilic molecules (citrate) have been suggested as directly contributing to toxicity of copper (32). Our observations do not appear to support this hypothesis. Hence, we contend that the enhanced metal availability inferred by Guy and Kean (32) in the presence of low molecular weight hydrophilic ligands may have been an artifact due to the use of an incorrect value of the metal citrate complexation constant. VOL. 36, NO. 14, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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It is important, yet difficult, to elucidate all sources of nitrification inhibition in the complex matrix of influent wastewater. By adding a sufficient amount of chelating agent to bind the free metal cations, a differentiation between metal and other sources of nitrification inhibition could be ascertained. Indeed, Wong et al. (33), using algal bioassays, have quantified the total toxicity and metal-associated toxicity in wastewater by adding EDTA. However, due to the potential inhibition from EDTA itself, as our results have shown, such data should be interpreted with caution. Alternatively, a noninhibitory chelating agent like NTA could be used to titrate metal-associated inhibition. In summary, the evaluation of inhibition of nitrifying activity by the heavy metal nickel and cadmium in batch assays yielded the following conclusions: (i) Nickel and cadmium inhibit ammonia oxidation but not nitrite oxidation at total analytical concentrations of approximately 1 mM. (ii) Inhibition by the heavy metal correlated well with the free cation concentration, [Ni2+] or [Cd2+], and not the total aqueous nickel or cadmium concentration, supporting the predictions from the FIAM. Accordingly, predictions and inhibition abatement should be based on free metal cation concentrations rather than on total metal concentrations. (iii) Addition of strong chelating agents (e.g., EDTA, NTA, citrate) can reduce the inhibitory effect of nickel or cadmium on nitrification, although at elevated dosages EDTA itself impairs nitrification kinetics. Remedial strategies for metal inhibition may be achieved by adding noninhibitory chelating agents.

Acknowledgments This study was funded, in part, by the Connecticut DEP through a U.S. EPA Long Island Sound Study grant under Section 119 of the Clean Water Act.

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Received for review November 9, 2001. Revised manuscript received April 8, 2002. Accepted April 22, 2002. ES015784A