Environ. Sci. Technol. 1996, 30, 2057-2065
Effect of Adsorption on the Biodegradation of Nitrilotriacetate by Chelatobacter heintzii H A R V E Y B O L T O N , J R . * ,† A N D DON C. GIRVIN‡ Environmental Microbiology and Interfacial Geochemistry Groups, Pacific Northwest National Laboratory, 900 Battelle Boulevard, Richland, Washington 99352
Nitrilotriacetic acid (NTA) is a synthetic chelating agent that was used to decontaminate nuclear reactors and was disposed at Department of Energy waste sites. NTA may influence the mobility and fate of radionuclides in soils and sediments if it is not biodegraded. Because it was unclear how adsorption of NTA would influence its degradation, experiments were conducted in a model system containing gibbsite and Chelatobacter heintzii with 60CoNTA at pH 7 and NTA at pH 6 and 8. The rates of NTA desorption from gibbsite were pH dependent (desorption half-lives at pH 6, 7, and 8 were 80, 16, and 1 h, respectively, while that of Co at pH 7 was 2.5 h for equal molar CoNTA). Degradation rates of NTA in solution by C. heintzii decreased as pH decreased and depended on the dominant form of NTA in solution (e.g., biodegradation rates for HNTA2- > AlOHNTA> CoNTA-). The degradation of NTA was significantly slower when NTA was adsorbed to gibbsite. This difference was observed at pH 6 and 8 with NTA and at pH 7 with NTA and equal molar cobalt. A coupled process model successfully simulated NTA desorption and degradation at all pHs. Both experimental data and simulations demonstrated that adsorbed NTA was unavailable for biodegradation and that the rate of desorption limited the rate of biodegradation. Only a fraction of the total 60Co (i.e., 4%) was associated with C. heintzii cells after NTA had been degraded in the presence of gibbsite. Therefore, the biodegradation of NTA by microorganisms similar to C. heintzii should not significantly alter 60Co sorption to aluminum oxides or 60Co transport where NTA and 60Co have been co-disposed.
* Corresponding author telephone: (509) 375-2758; fax: (509) 3756666; e-mail address: h
[email protected]. † Environmental Microbiology Group. ‡ Interfacial Geochemistry Group.
S0013-936X(95)00847-9 CCC: $12.00
1996 American Chemical Society
Introduction Multidentate synthetic chelating agents such as nitrilotriacetic acid (NTA) have been used to decontaminate nuclear and other materials (1, 2) and for processing nuclear waste (3). Synthetic chelating agents form stable watersoluble complexes with a wide range of radionuclides and metal ions (4, 5). The co-disposal of synthetic chelating agents with radionuclides has led to the contamination of groundwater with radionuclides (6-8). The enhanced mobility of cationic radionuclides in groundwater is presumably because of the altered adsorption of the radionuclides because they are chelated (4, 9). The adsorption of NTA to Fe (10) and aluminum oxides (11-13) increased as pH decreased (i.e., ligand-like adsorption), while adsorption of metals to aluminum and iron oxides decreased as pH decreased (i.e., cation-like adsorption). Adsorption of metals in the presence of chelating agents can be cation-like, ligand-like, or a combination of the two depending upon multiple equilibria among the metal, chelate, solid, solution pH, metal-to-chelate ratio, and solid-to-adsorbate ratio. In equal molar suspensions of Cu-, Ni-, Pb-, Zn-, and Cd-NTA, only Cu exhibited mixed cation-like and ligand-like adsorption on δ-Al2O3 (11). For the other metals, cation-like adsorption was suppressed by the NTA, and no ligand-like adsorption occurred. For equal molar Co and NTA in a gibbsite suspension, the adsorption of Co was predominantly cationlike with little ligand-like adsorption (13). Bacterial strains able to degrade NTA have been isolated from water (14, 15), subsoil (16), soil (17-20), and sewage effluent (21-24). Although there have been numerous studies of NTA degradation, there is currently only limited understanding of how adsorption and sorbent-induced changes in the aqueous speciation of NTA will influence its biodegradation. Metals, which alter the aqueous speciation of NTA, will influence NTA degradation (21, 25, 26). The aqueous speciation of NTA influenced its degradation by Chelatobacter heintzii (ATCC 29600) (26). The rate of NTA degradation was not related to the thermodynamic stability constants for various metal-NTA complexes, but it was related to the rates of formation of HNTA2- (26). Sorption of hydrophobic organic compounds by complex sorbents (e.g., soils, sediments, organic matter) will decrease their rate of biodegradation (27-30). Diffusion of the hydrophobic organic to the aqueous phase is often considered the rate-limiting step for degradation to occur (29). Adsorption of ionizable compounds will also limit biodegradation as has been shown for citrate (31), quinoline (30), phthalate, and salicylate (32). The adsorption of NTA did not appear to influence the long-term degradation of NTA in various subsurface sediments (33). However, this study did not quantify the populations of NTA degraders in the various sediments. It is therefore unclear if similar populations were present in sediments with varying amounts of sorbed NTA to adequately test whether sorption limited NTA degradation. The objective of the present study was to determine how the adsorption of NTA influenced its biodegradation. The hypothesis investigated was that adsorbed NTA is unavailable for degradation and that the rate of NTA desorption will limit the degradation rate. A well-
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characterized system was chosen for study including the sorbent gibbsite and C. heintzii, an NTA-degrading bacterium. Gibbsite is a dominant aluminum oxide in the environment (34), while C. heintzii has been wellcharacterized for the degradation of NTA (26). The rate of NTA degradation by C. heintzii should be influenced by both the rate of NTA desorption from gibbsite and the dissolution-induced changes in the aqueous speciation of NTA (12). Experimental data and a model linking NTA desorption and degradation were used to test the hypothesis.
Materials and Methods Radioisotopes and Radioactive Solutions. The following radioisotopes were used: 14C-labeled NTA (U-14C, 426 MBq mmol-1, 98% purity, Amersham, Arlington Heights, IL), 14Clabeled glucose (U-14C, 111 MBq mmol-1, 99% purity, NEN Research Products, Boston, MA), and 60Co (60CoCl2, 21090 MBq mg-1, 99% purity, NEN Research Products, Boston, MA). Radioactive solutions were prepared by mixing the above isotopes with Al, Co, stable NTA, stable glucose, and buffer as needed for specific experiments. The concentrations of Al and Co were measured by inductively coupled plasma-atom emission spectroscopy (ICP-AES). Unlabeled and 14C-labeled NTA were mixed to obtain 1 µM and ≈167 Bq mL-1. The ratio of 60Co to 59Co was 1:7300 when 60Co was used. The 14C or the 60Co-14C activity was determined by single label or dual-label scintillation counting, respectively. The 60Co activity was verified by γ-counting. Buffers. The buffers included pH 6.0 buffer [0.01 M piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) and 0.01 M NaNO3], pH 7.0 buffer [0.01 M N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) and 0.01 M NaNO3], and pH 8.0 buffer (0.01 M HEPES and 0.01 M NaNO3). These buffers do not compete with NTA as metal complexing agents (35, 36), do not enhance the dissolution of the gibbsite under experimental conditions used (data not shown), and do not alter the adsorption of Co or NTA to gibbsite at their respective pHs in comparison to 0.01 M NaClO4 (data not shown). Preparation of the Concentrated Gibbsite Suspension (CGS) Containing NTA and Co-NTA. Concentrated gibbsite suspensions containing NTA and Co-NTA were prepared so that greater than 99% of NTA was initially adsorbed to gibbsite. Gibbsite (R-Al(OH)3; Superfine 4, Alcan Chemicals, Alcan Aluminum Corp., Cleveland, OH) was treated with strong base and rinsed repeatedly with 0.01 M NaNO3 (13) prior to use. The measured N2 BET (Brunauer, Emmett, and Teller) surface area and mean particle diameter of the treated gibbsite were 3.5 m2 g-1 and 1.3 µm, respectively (13). Suspensions containing 7.5 g of gibbsite L-1 and 0.01 M NaNO3 were adjusted to pH 6, 7, and 8 and made to 0.01 M with the appropriate buffer; [14C]NTA or 60Co-[14C]NTA was added and allowed to mix overnight before the gibbsite was concentrated by repeated configuration. The gibbsite was then suspended in the original supernatant to ∼150 g of gibbsite L-1 to provide the concentrated gibbsite suspension (CGS). The concentration of Co and/or NTA in the solution phase of the CGS was determined by filtering (0.1 µm) an aliquot of the CGS and measuring the 14C or the 60Co-14C activity. The total concentration of Co and/or NTA in the CGS was determined by extracting with HNO3 (pH 3) for Co and with NaOH (pH 12.5) for NTA for 2 days, filtering (0.1 µm) the extract, and scintillation counting. Greater than 99% of the NTA or
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Co-NTA was adsorbed to the gibbsite in the CGS at all pHs. Desorption of NTA and Co-NTA from Gibbsite Using a Stir-Flow Cell. The rate of NTA and Co-NTA desorption from gibbsite was determined to model the desorption of NTA in the desorption-degradation experiment. The desorption of [14C]NTA at pH 6 and 8 or 60Co-[14C]NTA at pH 7 from gibbsite was investigated using a stir-flow cell (37, 38). Gibbsite was retained in the cell by a 0.1-µm filter as the influent buffer passed through the cell. The gibbsite suspension in the 9-mL cell was stirred at 2200 rpm to obtain ideal mixing or a homogeneous aqueous phase (39). The CGS was added to the cell, and flow was immediately started. The [14C]NTA or 60Co in the effluent was measured as a function of time. Desorption experiments were conducted for a range of flow rates (0.6-3.3 mL min-1) and gibbsite masses (0.05-0.96 g). Ideal mixing in the stir-flow cell (39) was tested in the absence of gibbsite by flowing 1 µM [14C]NTA through the cell until the effluent and influent concentration were equal, switching to an NTA-free influent solution, and measuring the decrease in radioactivity in the effluent solution. A similar experiment was performed in the presence of gibbsite (102 g L-1) using 1 µM pentafluorobenzoic acid (PFBA), which did not adsorb to gibbsite. The concentration of PFBA in the effluent was measured at 254 nm. Ideal mixing was demonstrated for all flow-cell conditions by using NTA and PFBA as tracers (39). Growth Conditions and Degradation of NTA. Chelatobacter heintzii (ATCC 29600) was grown with 5.23 mM NTA in a defined medium (16) at room temperature (22 °C) with shaking (150 rpm). Cells were harvested at late log phase by centrifuging at 8000g for 15 min and resuspended in either pH 6, 7, or 8 buffer and washed two more times with the respective buffer after centrifugation. All experiments contained approximately 108 colony forming units mL-1. For solution-degradation experiments (i.e., where all the organic is in solution), [14C]NTA, metal-[14C]NTA or [14C]glucose were added to the buffered cell suspensions. For the desorption-degradation experiments (i.e., where all the NTA was initially sorbed to gibbsite), approximately 1 mL of CGS containing adsorbed NTA or Co-NTA was added to buffered cell suspensions. Final solution volumes of 20 mL were contained in 200-mL acid-washed sterile glass bottles. A 14CO2 trap (1 mL of 0.3 M KOH in a 7-mL scintillation vial) was suspended in the headspace of the bottle, and the bottle was sealed with a Teflon-coated rubber stopper. The bottles were incubated with shaking (120 rpm) at ambient laboratory temperature (22 °C) and destructively sampled at several time intervals by acidifying to pH < 2 with HNO3 and allowing the 14CO2 to diffuse into the KOH trap for 48 h. Mineralization of NTA and glucose to 14CO2 was stopped after adjusting to pH 2. Three replicates were included for each sampling point. The traps were analyzed for 14C by liquid scintillation counting. Desorption-Degradation of NTA and Co-NTA. The desorption-degradation experiments at pH 6 and 8 for NTA and pH 7 for Co-NTA were started by adding the concentrated gibbsite suspension (CGS) to the buffered cell suspension. The concentration of NTA was 0.4 µM with greater than 99% of this adsorbed to 7.5 g of gibbsite L-1. Solution-Degradation of 1 µM NTA and 1 µM CoNTA. These experiments were designed to mimic the aqueous speciation of NTA in the desorption-degradation
experiments. The degradation of NTA-only (i.e., no metal added) was conducted at pH 6, 7, and 8 to determine the influence of pH on NTA degradation. Degradation of NTA in various Al-NTA systems was examined at pH 6, 7, and 8. The Al concentrations were based on the dissolved Al concentration in equilibrium with the gibbsite suspension at these pHs and included 1.1, 0.45, and 0.60 µM Al at pH 6, 7, and 8, respectively. Degradation of NTA in Co-NTA and Co-Al-NTA systems was examined at pH 7. The Co was added at 1 µM, while Al was added at 0.45 µM. Concentration Threshold for NTA Degradation. To ensure that C. heintzii was able to degrade the low-solution concentrations of NTA, which was expected in the desorption-degradation experiment at pH 6, degradation of NTA at 10-6-10-10 M was examined at pH 6. The degradation assay was modified by increasing the volume to 500 mL to provide enough 14C at the lowest concentration of NTA (10-10 M) to detect 14CO2. Evolution of 14CO2 at a single point (20 h) was determined. NTA Degradation with 60Co and 59Co. To determine if 60Co inhibited the degradation of NTA, separate degradation experiments were conducted with 1 µM 60Co-[14C]NTA and 59Co-[14C]NTA at pH 7. The treatments were sampled at 1, 8, and 24 h to determine 14CO2 evolved. Cobalt Binding to C. heintzii and Adsorption to Gibbsite during the Various Stages of Co-NTA Degradation at pH 7. The influence of NTA degradation on Co binding by C. heintzii cells and on Co sorption by gibbsite was investigated as a function of time. These studies investigated sorption of Co by gibbsite, binding of Co by C. heintzii, and both of these in a combined experiment. To determine Co sorption by gibbsite, Co-NTA adsorbed to CGS was added to buffer without cells. To determine the cellular binding of Co, equal molar Co-NTA (1 µM) was added to cell suspensions. Finally, to examine the competitive Co binding by cells and sorption by gibbsite, Co-NTA adsorbed to CGS was added to cell suspensions. The suspensions were sampled at 1.5, 6, and 30 h. A subsample (≈10 mL) was removed from the bottle, filtered through a 0.2-µm filter and weighed, and the 60Co in the filtrate was counted. The solution remaining in the bottle was acidified to determine 14CO2 evolved. Degradation of Glucose. The degradation of glucose was used to determine if the soluble Al concentrations used in the NTA solution-degradation experiments were inhibitory to C. heintzii. Glucose degradation was also used to determine if gibbsite inhibited the degradation of a nonsorbing organic. The initial concentration of glucose was 1 µM in all glucose-degradation experiments. The experiments were sampled as a function of time and included glucose-only at pH 6 and 8; glucose plus Al at pH 6 and 8 with total Al at 0.13 and 0.60 µM, respectively; and glucose plus 7.5 g gibbsite L-1 at pH 6 and 8. These Al concentrations were based on the dissolved Al concentrations in equilibrium with gibbsite at these pHs in the absence of NTA. Note that at pH 6 the Al concentration in the Al-glucose system was nearly a factor of 10 less than in the Al-NTA system (Al ) 1.1 µM). This enhancement is dissolved Al is due to the increase in gibbsite solubility by NTA. Concentrations of Al > 0.13 µM at pH 6 without NTA would exceed the gibbsite solubility limit, thus resulting in the possible precipitation of amorphous aluminum oxide. Controls of glucose plus 7.5 g of gibbsite L-1 at pH 6 and 8 without C. heintzii were included to measure glucose adsorption and degradation in the presence of only gibbsite.
The controls were sampled at 1 and 26 h. A 1-mL subsample was removed from the bottle and filtered through a 0.2-µm filter, and the 14C in the filtrate was counted. The solution remaining in the bottle was acidified, and the 14CO2 evolved was determined.
Modeling and Theory Aqueous Speciation Modeling. The distribution of NTA among its free acid and metal complex forms was calculated using the aqueous speciation-solubility model MINTEQ and its associated data base (40). These calculations were based on the concentrations of cations (ICP-AES), anions, and NTA; measured pH; and the thermodynamic stability constants for H3NTA and metal-NTA complexes (5, 4042). Discussions of the dominant metal-NTA species or the NTA speciation refers to the species distribution as calculated using MINTEQ. Biodegradation 14CO2 Modeling. Biodegradation of NTA in solution was a first-order process at NTA concentrations from 0.05 to 5.23 µM (26) and is represented here by the reaction:
C9 8 CO2 k
(1)
deg
where C is the concentration of NTA in solution and CO2 is from the degradation of NTA. A pseudo-first-order product curve
P ) Po(1 - exp-kdegt)
(2)
modeled the 14CO2 evolved (P) as a function of time (t) and provided the first-order rate constant (kdeg) and the asymptote (Po) (43). The rate constant and asymptote and their 95% confidence intervals were estimated by nonlinear least-squares regression using quasi-Newton estimation (44). The half-life for NTA degradation to CO2 was calculated from
t1/2 )
ln 2 kdeg
(3)
Two-State Adsorption-Desorption Model. The model describing the adsorption-desorption of NTA by gibbsite assumes that NTA on the surface can occupy two distinct configurations or states, S1 and S2, with S2 being an intermediate state in the NTA adsorption-desorption processes. This is depicted by k1b
k2b
1f
2f
S1 {\ } S2 {\ }C k k
(4)
where Si is the adsorbed NTA concentration (mol g-1), C is the solution NTA concentration (mol L-1), and kif and kib (h-1) are the forward and reverse rate constants for i ) 1, 2. The distribution coefficient at equilibrium (Kd) is
Kd )
(S1 + S2) V ) Keq C m
(5)
where Kd has units of L g-1, Keq is a unitless adsorption equilibrium constant, and m and V are the sorbent mass (g) and solution volume (L), respectively. Utilizing the principal of microreversibility (45), Keq can be written in terms of the forward and reverse rate constants:
Keq )
( )
k2f k1fk2f + k2b k1bk2b
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(6)
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Stir-Flow Cell. Based on the mass balance expression for an ideally mixed stir-flow cell (37), the concentration of aqueous NTA in the stir-flow cell and the cell effluent are identical and can be given by
dC Q m ) (Cin - C) - k2fC - k2bS2 dt V V
(
)
(7)
where Q is the flow rate (L h-1) through the cell, V is the cell solution volume (L), Cin is the influent concentration of NTA, C is the effluent concentration of NTA, m is the mass of gibbsite in the cell (g), and t is time (h). The firstorder rate expressions for the two-state adsorption model are
dS2 V + k1bS1 - k1fS2 ) -k2bS2 + k2fC dt m
(8)
dS1 ) -k1bS1 + k1fS2 dt
(9)
( )
For the desorption experiments, Cin ) 0 and the initial conditions at t ) 0 were C ) S2 ) 0 and S1 ) the total initial adsorbed concentration of NTA or Co-NTA. Coupled Sorption-Degradation Model. The coupled sorption-degradation model is an extension of the twostate adsorption model in that only NTA in solution is degraded. This is described by the series of reactions k1b
k2b
1f
2f
kdeg
} S2′ {\ } C′ 98 CO2 S1′ {\ k k
(10)
where S1′, S2′, and C′ are dimensionless concentrations (fraction of the total NTA) in the sorbed states and in solution, respectively, and CO2 is the fraction of the total NTA-C transformed to CO2. The linear first-order rate equations describing the coupled sorption-degradation process are
dS1′ ) -k1bS1′ + k1fS2′ dt
(11)
dS2′ ) -k1fS2′ + k1bS1′ - k2bS2′ + k2fC′ dt
(12)
dC′ ) k2bS2′ - k2fC′ - kdegC′ dt
(13)
dCO2 ) RkdegC′ dt
(14)
For the desorption-degradation experiments, the initial conditions at t ) 0 were identical to those in the desorption experiments, that is C′ ) S2′ ) 0 and S1′ ) 1 ()the total initial adsorbed concentration of NTA or Co-NTA). The R in eq 14 is a correction factor to account for the incomplete conversion of NTA-C to CO2. The R was the calculated Po (i.e., percent CO2 evolved)/100 for the solution experiment whose aqueous speciation was that expected in the desorption-degradation experiment. The system of equations describing desorption experiments (eqs 7-9) and desorption-degradation experiments (eqs 11-14) were solved numerically using a Runge-Kutta algorithm.
Results and Discussion Solution Degradation of NTA. An understanding of how pH, aqueous speciation, and metal toxicity influenced the
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FIGURE 1. Degradation of 1 µM NTA as a function of time at pH 6, 7, and 8; NTA plus Al at pH 6 (1 µM Al), 7 (0.45 µM Al), and 8 (0.60 µM Al); NTA plus 1 µM Co at pH 7; and NTA plus 1 µM Co and 0.45 µM Al at pH 7.
degradation of NTA in solution was necessary before we could interpret and model the degradation of NTA initially adsorbed to gibbsite. The rate of NTA degradation with no metal added to solution (NTA-only) decreased as pH decreased (Figure 1) with degradation complete after 5 h for pH 8 and approaching the asymptote at 32 h for pH 6. The dominant aqueous species was HNTA2- over this pH range. The rate constants for the degradation of NTA-only differed over this pH range (Table 1), but the asymptotes were the same (data not shown). This suggested that pH had either a general effect on microbial metabolism and the generation of 14CO2 or a direct effect on the degradation on NTA. The degradation of glucose was the same at pH 6 and 8 (Figure 2), suggesting that the decrease in the degradation of NTA as a function of pH (Figure 1) was specific for NTA. This pH dependence of NTA degradation may be due to a change in the structure of HNTA2- as a function of pH. Nuclear magnetic resonance showed that the N-protonated isomer of HNTA2- dominates in solution at pH 10, while the carboxylate-protonated isomer dominates at pH 5 (46). This change in the location of the proton on NTA as a function of pH alters its configuration and charge distribution. This may affect the transport of NTA into C. heintzii and therefore the rate of degradation. The rate of NTA degradation decreased for the Al-NTA system as pH decreased (Figure 1, Table 1). The rates of
TABLE 1
TABLE 2
First-Order Degradation and Sorption Rate Constants (h-1), Equilibrium Sorption Parameters, and Initial NTA Concentration in Stir-Flow Cell at pH 6, 7, and 8
Summary of Equilibrium Species Distribution of 1 µM NTA for Solution-Degradation Experimentsa
constants
kNTA-onlya kAl-NTA kCo-NTA kCo-Al-NTA k2fc k2b k1f k1b Keqd solid/solutione Kdf initial nM NTA in cell
pH 8
pH 7
pH 6
2.0 2.1 ndb nd 2.7 1.5 0.12 3.5 1.86 7.5 0.25 2.8
0.40 0.14 0.125 0.133 2.8 0.165 0.12 0.16 30 93.4 0.32 40.3
0.12 0.033 nd nd 5 0.04 0.12 0.09 291 107 2.71 71.9
a
Biodegradation rate constants determined from data in Figure 1. b Not determined (nd). c Desorption rate constants determined from data in Figure 3. d Adsorption constant at equilibrium. e Solid to solution ratio (g L-1) used in the flow cell for the desorption experiments. f Measured distribution coefficient at equilibrium (L g-1).
FIGURE 2. Degradation of 1 µM glucose as a function of time including glucose-only at pH 6 and 8, glucose plus Al at pH 6 (0.13 µM Al) and 8 (0.60 µM Al), and glucose plus gibbsite at pH 6 and 8.
degradation in the Al-NTA and NTA-only systems were identical at pH 8 (Figure 1, Table 1) presumably because HNTA2- was the dominant species in both systems (Table 2). As pH decreased, the rate of NTA degradation in the Al-NTA system decreased relative to the NTA-only system and AlOHNTA- and AlNTA0 replaced HNTA2- as the dominant NTA species (Table 2). This suggested that HNTA2- was more degradable than Al-NTA complexes. However, Al toxicity may have contributed to the decreased rates of degradation found in the Al-NTA versus HNTA2systems at pH 6. Degradation of glucose at pH 6 was reduced when Al (0.13 µM) was added, while at pH 8 (Al ) 0.6 µM) there was no Al inhibition (Figure 2). These Al concentrations were based on the dissolved concentrations of Al in equilibrium with gibbsite at pH 6 and 8 in the absence of NTA. The
system
pH
NTA-only
6 7 8 6
Al-NTA
7 8 Co-NTA Co-Al-NTA
7 7
NTA solution species distribution (% of total NTA) HNTA2- (99%) HNTA2- (99%) HNTA2- (99%) AlOHNTA- (74%), AlNTA (15%), HNTA2- (10%), Al(OH)2NTA2- (1%) HNTA2- (65%), AlOHNTA- (30%), Al(OH)2NTA2- (5%) HNTA2- (90%), Al(OH)2NTA2- (7%), AlOHNTA- (3%) CoNTA- (96%), HNTA2- (4%) CoNTA- (86%), AlOHNTA- (7.5%), HNTA2- (5.5%), Al(OH)2NTA2 (1%)
a Al was added at 1.1, 0.45, and 0.6 µM for pH 6, 7, and 8, respectively. Co was added at 1 µM.
greater inhibition of glucose degradation by Al at pH 6 than 8, even though there was less total Al, may have been because of the much larger concentration of Al3+ (106 times greater than at pH 8). The ionic form of metals (e.g., Al3+) is usually more toxic than the hydrolyzed (e.g., Al(OH)2+) or complexed forms (AlOHNTA-) (47). The inhibition of glucose degradation at pH 6 by Al suggested that a portion of the decrease in NTA degradation at pH 6 may have been because of Al3+ toxicity. However, the greater inhibition of Al on the degradation of NTA (Figure 1) than glucose (Figure 2) indicated that changes in NTA speciation from HNTA2- to Al-NTA species (Table 2) was also responsible for the significant decrease in NTA degradation. The rate of 1 µM NTA degradation at pH 7 decreased in the following order: NTA-only > Al-NTA (0.45 µM Al) > Co-NTA (1 µM Co) ) Co-Al-NTA (1 µM Co, 0.45 µM Al) (Figure 1, Table 1). The predominance of HNTA2- followed the same relative order, with 100, 65, 4, and 6% of the NTA as HNTA2- in these systems (Table 2). The dominant species of NTA in the Co-NTA and Co-Al-NTA systems was CoNTA- (96 and 86%, respectively). This was consistent with the observation that the degradation of NTA by C. heintzii was strongly dependent on the chelated metal (26). This previous study also determined that toxicity of Co did not influence the degradation of NTA (26). However, the previous study used stable 59Co, while the present study used 60Co. Similar rates of NTA degradation were found when either 60Co or 59Co were present (data not shown), demonstrating that 60Co did not inhibit the degradation of NTA. Thus, the difference in the degradation of NTA in these solution systems was predominantly due to the differences in the aqueous speciation of NTA with degradation of NTA following the order: HNTA2- > Al-NTA species > CoNTA-. The rates of NTA degradation determined here in solution (Table 2) were used to simulate degradation in the desorption-degradation studies. Desorption of NTA from Gibbsite. An understanding of how pH influenced the desorption of NTA from gibbsite was necessary before we could interpret and simulate the degradation of NTA initially adsorbed to gibbsite. The profiles for the desorption of NTA at pH 8, 7, and 6 and Co at pH 7 are shown in Figure 3. These profiles have been corrected for the washout of the NTA in the CGS, which was initially in solution (
