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Applied and Environmental Microbiology, October 2005, p. 5888-5892, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5888-5892.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Improvement of NaNO₂-Oxidizing Activity in Nitrobacter vulgaris by Coentrapment in Polyacrylamide Containing Polydimethylsiloxane Copolymer and DEAE-Sephadex

Songping Zhang,^1,3 Olof Norrlöw,² and Estera Szwajcer Dey^1*

Pure and Applied Biochemistry, Lund University, Box 124, 221 00 Lund, Sweden,¹ Recycling Competence Centre, Kemira Kemi AB, Box 902, 251 09 Helsinborg, Sweden,² National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, People's Republic of China³

Received 26 February 2005/ Accepted 29 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Removal of nitrite and nitrate from drinking water has attracted great attention in recent years because of the human health risk induced by the exposure to contaminated groundwater and surface water. We have therefore tested a model nitrite oxidation system by coentrapping the NaNO₂ oxidizer Nitrobacter vulgaris with polydimethylsiloxane (PDMS) copolymer and DEAE-Sephadex in a polyacrylamide gel. The copolymer and the anion exchanger facilitate the diffusion of oxygen and NaNO₂, respectively, into the gel matrix. To test the nitrite-oxidizing activity, the entrapped cells were coupled to a thermal sensor. Coentrapment of 5% (wt/vol) DEAE-Sephadex with Nitrobacter vulgaris increased the nitrite-oxidizing activity by a factor of 3.7 compared to entrapped cells alone, and by the addition of 0.86% (wt/vol) artificial oxygen carrier PDMS copolymer increased the activity further to 4.3 times higher. Operational and storage stability of the coentrapped N.vulgaris also improved. This suggests that this enhanced immobilized cell system can also be used for nitrite oxidation to nitrate in drinking water as an on-line thermally monitored bioreactor.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The removal of nitrate (NO₃^–) and nitrite (NO₂^–) from drinking water has attracted great attention in recent years because of the human health risk caused by the exposure to groundwater and surface water contaminated with these anions. This risk is due to NaNO₂'s direct toxicity by causing methemoglobinemia and cancer (18). The NaNO₂ concentration permitted in drinking water is 0.2 mg liter^–1 (30). Biological ammonia removal occurs by the process of nitrification to nitrate. Nitrate can subsequently be denitrified to nitrogen. Chemo-litho-autotrophic bacteria oxidize ammonia to nitrate in two separate steps. The oxidation of ammonia to nitrite is carried out by Nitrosomonas, Nitrosospira, Nitrosovibrio, and Nitrosococcus, while nitrite oxidizers include Nitrobacter, Nitrospira, Nitrococcus, and Nitrospina gracilis species (2, 4). In denitrification after NaNO₂ oxidation, nitrate is converted by denitrifying bacteria under anaerobic conditions to nitrogen gas. The nitrification steps often become rate limited by oxygen availability. The oxidation of NaNO₂ is catalyzed by nitrite oxidoreductase, which is located at the inner side of the cytoplasmic and intracytoplasmic membranes of Nitrobacter cells (29, 36). NaNO₂ oxidizers are slow-growing organisms with generation times of up to 140 h (3), which makes them easily washed out from treatment plants (23). They are therefore rarely responsible for nitrite oxidation in wastewater treatment. Nitrifiers can be immobilized in a polymer matrix (1, 10, 15, 19, 32-35, 38, 39) or attached to a membrane with a micro- or macroscale (27). The immobilized nitrifiers (28) are already used in continuously operating pilot units for nitrogen removal from different wastewater sources in the Czech Republic, Chemopetrol a.s. Litvinov. The main advantage of immobilized cells is the high concentration of active biocatalyst attainable in the solid phase (10). The disadvantage is that in such high-density reactors the conversion rate can be limited by the diffusion of substrates, i.e., oxygen and NaNO₂ (38).

To solve the problem of oxygen limitation, pure oxygen can be used instead of air, leading to better oxygen mass transfer and saturation. The drawback of using pure oxygen is that it is expensive. Another option is to increase the oxygen-carrying capacity of the aerobic reaction system by using oxygen carriers. So far, a variety of oxygen carriers have been employed in biological systems, such as hemoglobin (26), perfluorochemicals (12), dodecane (17), and polydimethylsiloxane (PDMS) or PDMS copolymer (11, 20). PDMS oil and PDMS copolymer successfully increased the solubility of oxygen in the tested biological systems (11, 37). Apart from oxygen carriers, catalase was also used as an oxygen generator in the presence of H₂O₂ (7, 14) by catalyzing the reaction (24) shown in equation 1:

Biological reactions are always associated with heat gain or loss. Even though microbial metabolism is composed of many individual reactions, the net conversion process can be thermodynamically treated as a simple chemical reaction. Therefore, the net metabolic activity of living cells can be evaluated by calorimetry. The conversion of NaNO₂ to nitrate carried out by nitrite oxidoreductase can be described by equation 2 (9): The heat production rate (electrical signal) upon the injection of NaNO₂ substrate solution to the immobilized N. vulgaris-based microbial thermosensor system (6) measures NaNO₂ oxidoreductase activity of the N. vulgaris cells. Of three Nitrobacter sp. strains used for nitrite analysis (without enhancer), Nitrobacter vulgaris was shown to be most suitable for the construction of microbial biosensor (25).

The objective of these studies was to show that an improved NaNO₂-oxidizing activity of a laboratory strain of N. vulgaris could be attained when cells were coimmobilized with an anion exchanger and an oxygen carrier. This suggests that an analogous bioreactor can easily be constructed to remove NO₂^– from drinking water. We also show that the microbial thermosensor can be used for quantitative determination of nitrite conversion to nitrate.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials.
Acrylamide/bis-acrylamide (19:1 [wt/wt]) from Sigma (St. Louis, MO) was prepared as a 40% stock solution. N,N,N',N'-tetramethyethylenediamine (TEMED) and ammonium persulfate [(NH₄)₂S₂O₈] were from Fluka (Buch, Switzerland). DEAE-Sephadex A-25 was purchased from Amersham Biosciences AB (Uppsala, Sweden). PDMS copolymer Q2-5247 fluid, composed of 18% dimethylsiloxane, 35% ethylene oxide, and 46% propylene oxide, was a gift from Dow Corning (Midland, MI). Catalase (catazyme 25L; 25,000 U/ml) from Aspergillus niger was provided by Novo Nordisk (Bagsvaerd, Denmark). Trisoperl controlled-pore glass beads (CPG; particle diameter, 125 to 140 µm; pore diameter, 49.6 nm) with free amino groups were from Schuller GmbH (Steinach, Germany).

All other chemicals were of analytical grade and purchased from various commercial sources.

Bacterial strain and culture maintenance.
Nitrobacter vulgaris (DSM 10236) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) as an actively growing culture. Propagation was carried out by inoculating 2 ml of the received culture to 200 ml mixotrophic medium and incubating the mixture on a rotary shaker (150 rpm) in the dark at 28°C until the attenuance at 450 nm (D₄₅₀) had reached about 0.4. Nitrobacter vulgaris was grown in a modification of the mixotrophic medium described by Bock et al. (3). Two grams of NaNO₂ instead of 1.0 g was used, and 1.0 ml of trace element solution per liter was included. The trace element solution had the following composition (per liter): 33.8 mg MnSO₄·H₂O, 49.4 mg H₃BO₃, 43.1 mg ZnSO₄·7H₂O, 37.1 mg (NH₄)₆Mo₇O₂₄, 97.3 mg FeSO₄·7H₂O, 25.0 mg CuSO₄·5H₂O. The pH of the medium was adjusted to 7.4 with NaOH. The mixotrophic medium was selected instead of the heterotrophic because cells cultured in the former medium grow better, according to Bock et al. (3).

The growth of the cells was monitored by measuring the D₄₅₀ using a HITACHI U-3200 spectrophotometer and the NaNO₂ consumption rate using a Merck nitrite Spectroquant test kit (no. 1.14547.0001). When NaNO₂ in the medium was depleted, concentrated NaNO₂ solution (100 g liter^–1) was added to a final concentration of 2.0 g liter^–1. For preparation of frozen inoculates, 180ml of cell suspension (D₄₅₀ = 0.37) was harvested by centrifugation at +4°C for 15 min at 10,000 x g and then resuspended in 60 ml mixotrophic medium supplemented with 5% dimethyl sulfoxide as cryoprotectant. The cell suspension was distributed to 1.5-ml cryovials, frozen in liquid nitrogen for 30 min, and then stored in a –80°C freezer until use. For inoculation, one or two vials of inocula were used.

Entrapment of N. vulgaris in polyacrylamide gel.
Phosphate buffer (100 mM, pH 7.0) was used through the entrapment procedure. A 20% (wt/vol) acrylamide/bis-acrylamide stock solution (19:1 [wt/wt]) in phosphate buffer was prepared and stored at +4°C. One hundred milliliters of cell suspension at a D₄₅₀ of 0.74 was harvested by centrifugation as above. After subsequent washing with 60 ml 0.9% NaCl solution, the pellet was resuspended in 2.5 ml cold phosphate buffer (100 mM, pH 7.0) and used for entrapment.

Four kinds of additives were coentrapped with N. vulgaris cells in polyacrylamide gel (Table 1). A cell suspension (2.5 ml) of N. vulgaris was entrapped by stirring at low speed with acrylamide/bis-acrylamide (5 ml, 20%) and different additives (Table 1). Ammonium persulfate and TEMED were then added under stirring to initiate the polymerization of acrylamide. Ten milliliters of each gel mixture was poured into a petri dish (8.5 cm diameter) and kept at room temperature for 1 h and then stored at +4°C overnight. The thickness of the gel was measured as 1.7 mm. The gel was then pressed through a stainless steel sieve of 1-mm mesh. This procedure results in rod-shaped gel particles with a smooth surface. The gel particles were washed with 130 ml phosphate buffer by filtration through a funnel and kept in phosphate buffer at +4°C.

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TABLE 1. Compositions of polyacrylamide gels

Four kinds of polyacrylamide gel beads were prepared (Table 1): (i) gel with entrapped N. vulgaris cells only (G-1); (ii) gel with cells coentrapped with CPG-catalase (G-2); (iii) gel with cells coentrapped with 5% (wt/vol) anion exchanger DEAE-Sephadex (G-3); and (iv) gel with cells coentrapped with 5% (wt/vol) DEAE-Sephadex and 5% (wt/vol) PDMS copolymer Q2-5247 (G-4).

Determination of the entrapped amount of PDMS copolymer.
During the washing procedure, part of the PDMS copolymer was washed out from the gel matrix of G-4. To make a reference sample, 0.5 ml of PDMS copolymer (the same amount as used for entrapment) was mixed with 129.5 ml phosphate buffer to get the same volume of washing buffer. Si elemental concentration was analyzed by inductively coupled plasma-optical emission spectroscopy with an ICP-OES Optima 4300V apparatus from Perkin-Elmer. The results showed that Si elemental concentrations in the reference sample and washing buffer solutions were 580 and 480 mg Si kg^–1 solution, respectively. The initial PDMS copolymer concentration in the mixture before polymerization was 5% (wt/vol) (Table 1). Therefore, the actual concentration of PDMS copolymer entrapped in G-4 was calculated from the equation % PDMS = (1 – 480/580) x 5%, or 0.86%.

MT testing.
The polyacrylamide gel beads G-1, G-2, G-3, and G-4 were transferred into a column (0.7 ml; dimensions of 2.5 by 0.7 cm) and assembled with the thermometric system to construct a microbial thermosensor (MT). The MT unit was maintained at 30°C by an outer jacket (8). The nitrite-oxidizing activity of the immobilized N. vulgaris was tested under the following experimental conditions: (i) phosphate buffer (50 mM, pH 7.8) as running buffer (except when indicated) at a constant flow rate of 0.7 ml min^–1. (ii) NaNO₂ solutions of defined concentration were prepared in running buffer, and 0.5 ml was injected into the MT unit. The reaction velocity was determined from the peak height representing heat produced during the enzymatic oxidation of NaNO₂ (6).

(i) Testing of coentrapped CPG-catalase as oxygen supplier.
A series of MT testing was done on a biosensor consisting of coentrapped N. vulgaris and CPG-catalase (G-2). The signals from injection of 0.5-ml samples containing either NaNO₂ (145 mM) or H₂O₂ (2.94 mM) or a mixture were recorded and compared. In another experimental procedure, H₂O₂ (2.94 mM) was continuously pumped through the MT unit. After 10 min, when a constant signal from the enzymatic reaction between CPG-catalase and H₂O₂ was obtained, NaNO₂ (0.5ml, 145 mM) was then injected. The injection of NaNO₂ was repeated when the electrical signal returned to a constant value.

(ii) Calibration curves for NaNO₂.
NaNO₂ solutions (0.5 ml) with concentrations ranging from 0 to 261 mM were used to calibrate the NaNO₂ MT, consisting of G-1, G-3, or G-4 (Table 1). Calibration curves for each MT were obtained by plotting the electrical signal (peak height) against various NaNO₂ concentrations.

(iii) Preparation of solutions for measurement of the effect of aeration conditions on nitrite-oxidizing activity.
The running buffer and NaNO₂ solutions used were degassed by sonication under vacuum for 30 min. The MT unit was equilibrated with the degassed buffer for 20 min followed by injection of the degassed substrate. Oxygen-saturated buffer and NaNO₂ solutions were made with oxygen by bubbling with pure oxygen for 20 min. For each condition, the experiment was carried out in triplicate.

To determine if the observed nitrifying activities were of statistical significance under degassed and oxygen-aerated conditions, a standard t test was performed, proper for small population samples (22). The null hypothesis was assumed, i.e., that there is no difference under diverse aeration conditions. From the average signal values, the standard error, and the degrees of freedom, which was 4, the t values and the probability P values were calculated. The P values were estimated by applying the software SigmaStat (version 3.01 A; SYSTAT). When P is less than 0.05, it can be concluded that there are significant differences.

(iv) Effect of pH on nitrite-oxidizing activity.
Nitrite-oxidizing activity assays with MTs G-1, G-3, and G-4 were carried out over a pH range of 6 to 9. Phosphate (50 mM) and Tris-HCl (50 mM) buffers were used for pH ranges of 6.0 to 8.0 and 8.5 to 9.0, respectively. For each new pH, the MT unit was equilibrated with the new running buffer for at least 30 min at the flow rate of 0.7 ml min^–1 before NaNO₂ sample injection.

(v) Operational and storage stability.
The operational stability of MT was tested with G-4 as an example. Within 1 day, 20 successive injections were carried out. The storage stabilities of G-1, G-3, and G-4 were tested by measuring the nitrite-oxidizing activity for a period of 60 days. The columns were stored in phosphate buffer (50 mM, pH 7.8) at +4°C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Testing of coentrapped catalase as oxygen supplier.
Figure 1 illustrates the thermosensor testing of an MT consisting of coentrapped N. vulgaris cells with CPG-catalase (G-2). Peak a shows the signal from injection of NaNO₂ alone. H₂O₂ gave a 4-times-higher signal (peak b), while the signal from solution containing both NaNO₂ and H₂O₂ was 2.5 times higher than control (c). This was unexpected, since a larger signal is supposed to result from the combined two enzymatic reactions, hydrogen peroxide oxido-reduction and nitrification. Continuous supply of H₂O₂ resulted in a constant signal similar to that of b (d). Nevertheless, a large decrease in the signal (shown as a negative peak) subsequently appeared after injection of NaNO₂ (e). Continued supply of H₂O₂ increased the electrical signal but to a lower constant value. Repeated injection of NaNO₂ resulted in a new negative peak, which again returned to another even-lower constant signal (f).

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FIG. 1. Testing of Nitrobacter coentrapped with CPG-catalase (G-2) as a microbial thermosensor. Electrical signals shown resulted from 0.5 ml of 145 mM NaNO2 (a), 0.5 ml of 2.94 mM H2O2 (b), 0.5 ml of 2.94 mM H2O2 and 145 mM NaNO2 (c), continuous loading of 2.94 mM H2O2 (d), injection of 0.5 ml 145 mM NaNO2 (e), or repeated injection of 0.5 ml 145 mM NaNO2 (f).

Calibration curves for NaNO₂ from MT testing.
Figure 2 shows the typical recording from NaNO₂ with MTs G-1, G-3, and G-4. With MTs G-3 and G-4, a symmetric positive peak followed by a negative peak was recorded (Fig. 2).

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FIG. 2. A typical electrical signal versus time recordings of a nitrite microbial thermosensor consisting of entrapped N. vulgaris alone (G-1), N. vulgaris coentrapped with DEAE-Sephadex (G-3), or N.vulgaris coentrapped with DEAE-Sephadex and PDMS copolymer (G-4). Running buffer was phosphate buffer (0.05 M; pH 7.8). NaNO2 concentration was 87 mM.

Figure 3 compares calibration plots for NaNO₂ obtained with MT G-1, G-3, and G-4 gels. A good linear relationship between electrical signal and NaNO₂ concentration up to 200 mM was observed. For the MTs G-1, G-3, and G-4, the slopes of the calibration curves are 0.183, 0.671, and 0.790. The slope is thus a measure of their nitrite-oxidizing activity. Compared to the nitrite-oxidizing activity of gel with cells alone (G-1), the activity of G-3 is 3.7 times higher. When 0.86% PDMS copolymer and DEAE-Sephadex (G-4) were coentrapped, the nitrite-oxidizing activity was 4.3 times higher.

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FIG. 3. Calibration curves for nitrite from microbial thermosensors consisting of G-1 (; y = 0.183x; R2 = 0.997), G-3 (; y = 0.671x; R2=0.972), and G-4 (; y = 0.790x; R2 = 0.991). Linear regression analysis was done for NaNO2 concentrations ranging from 0 to 200 mM. Phosphate buffer (50 mM; pH 7.8) was used as running buffer.

Effect of oxygen saturation on nitrite-oxidizing activity.
The effects of oxygen on nitrite-oxidizing activity of the MTs G-3 and G-4 are illustrated in Table 2. The significance test results shown in Table 2 suggest that for the MT G-4, the electrical signal from oxygen-saturated NaNO₂ and running buffer increased 20 to 23% compared to the signal in the degassed system with the tested NaNO₂ concentrations, and the difference between them was statistically significant (P values are 0.0154 and 0.0009 under NaNO₂ concentrations of 7.22 mM and 14.5 mM, respectively). The biosensor consisting of G-3 without PDMS showed no significant increase (P values are 0.656 and 0.223 under NaNO₂ concentrations of 7.22 mM and 14.5 mM, respectively) under oxygen-saturated conditions.

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TABLE 2. Effects of aeration conditions on nitrite-oxidizing activity of the MTs G-3 and G-4a

Effect of pH on nitrite-oxidizing activity.
The NaNO₂ biosensors were also characterized with respect to pH. For G-3 and G-4, the highest nitrite-oxidizing activity was obtained at pH 8.0, which is in agreement with results obtained with free Nitrobacter cells (13).

Operational reproducibility and storage stability.
The MT G-4 biosensor was used for a repeated run of experiments. During 20 successive runs, very little fluctuation in activity relative to the first run was observed. The average peak height was 12.12 cm, and the standard deviation was 0.014. After 60 days refrigeration at 4°C, the MT G-4 retained nitrite-oxidizing activity as high as 80%. MTs G-1 and G-3, however, retained 64% and 63%, respectively (data not shown).

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have enhanced the nitrite-oxidizing activity of entrapped N. vulgaris cells by coentrapping oxygen-providing materials and promoting NaNO₂ diffusion to the cells. In previous experiments, catalase was used to provide oxygen from H₂O₂ (14). Earlier reports have shown that coimmobilized enzyme systems give a higher efficiency and sensitivity and faster response time than immobilized enzymes alone (21). To provide oxygen for nitrite oxidation, we have coentrapped CPG-catalase and N. vulgaris. Injection of H₂O₂ and NaNO₂ gave a lower response than that from H₂O₂ alone (Fig. 1, peaks b and c). Therefore, NaNO₂ may have an inhibitory effect on catalase activity. This was confirmed by the large negative signal induced by injection of NaNO₂ with continuous H₂O₂. The subsequent recovery from inhibition suggests a reversible NaNO₂-catalase inhibition (Fig. 1, e and f). Previous researches also showed that catalase (31) and other ferriheme-containing enzymes (40) were inhibited by millimolar NaNO₂. Consequently, catalase or hemoglobin (data not shown) was not as efficient as an oxygen source. We therefore selected the PDMS copolymer Q2-5247 (11). With immobilized cells, the gel matrix becomes a barrier to the mass transfer of both oxygen and NaNO₂. Coentrapment of the anion exchanger shown here is to facilitate the permeation of NaNO₂ into the gel. Thus, with the G-3 gel the signal was 3.7 times more than from gel G-1 (Fig. 2 and 3). Based on our results, we can speculate that the NO₃^– formed by oxidation competes with and replaces the adsorbed NO₂^– from DEAE-Sephadex. Ion exchangers have also been used for the removal of ammonia from wastewater (16). DEAE Sephadex has no oxygen carrier capacity, while PDMS copolymer does (Table 2).

PDMS copolymer in the gel G-4 further increased nitrite-oxidizing activity. It was not feasible to use higher concentration of PDMS copolymer, because this inhibited the polymerization process used. Previous research in the lab showed that at least 15% copolymer was needed to improve glucose oxidase activity (11). Figure 3 showed the rate of catalysis rose linearly as NaNO₂ increased up to 200 mM and then leveled off. This behavior is in agreement with Michaelis-Menten kinetics.

Coimmobilization of PDMS copolymer also improved the storage stability over a period of 60 days. A long operational and storage stability is a very important property for practical biosensor applications and in continuous wastewater treatment plants. The half time for the immobilized cell activity in Nitrosomonas europaea immobilized with calcium alginate was only 10 day at +4°C (33).

We have achieved the goal of this work, which was to enhance the nitrification by N. vulgaris coimmobilized in polyacrylamide with PDMS copolymer and DEAE-Sephadex. We demonstrated this by using the flow injection analytical scale system assisted by thermal measurements. Gel G-4 can now also be used in a scaled-up system in the form of a bioreactor for a continuous conversion of toxic NaNO₂ to less-toxic nitrate, with on-line thermal monitoring. Of three Nitrobacter spp. strains used for nitrite analysis (without enhancer), Nitrobacter vulgaris was shown to be most suitable (25). Mixed populations of immobilized nitrifiers have been widely used for cleaning of different types of wastewaters, but nitrification was not found to be enhanced by any additional agents (28), in contrast to our present work. In the Czech Republic, Chemopetrol A.S. Litvinov operates pilot units for nitrogen removal with encapsulated nitrifiers using LentiKats technology, but also without additives, for improving their performance.

 
We thank Kemira Kemi AB for financial support, Bengt Danielsson for permission to use the enzyme thermistor, and Simon Gough for critical reading of the manuscript. We are also grateful to Dow Corning for providing us with the PDMS copolymer (Q2-5247 fluid).

 
* Corresponding author. Mailing address: Pure and Applied Biochemistry, Lund University, Box 124, 221 00 Lund, Sweden. Phone: 46 46 2228258. Fax: 46 46 2224611. E-mail: estera.dey{at}tbiokem.lth.se.

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Applied and Environmental Microbiology, October 2005, p. 5888-5892, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5888-5892.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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