Behavior of dissolved organic carbon sources on the microbial reduction and precipitation of vanadium(V) in groundwater

Abstract

The performance of anaerobic microbial vanadium(V) reduction using five ordinary dissolved organic carbon sources was evaluated. In general, V(V) removal efficiency decreased with an increase in the molecular weight of the carbon substrate. In addition, organic acids supported a higher V(V) removal than alcohols, thus achieving the highest V(V) removal efficiency of 75.6% using acetate during a 12 h operation, compared with lactate, glucose, citrate and soluble starch. A higher initial V(V) concentration led to a lower V(V) removal efficiency, while the extra addition of organics had little effect on its improvement. With an increase in the pH and conductivity, the V(V) removal efficiency first increased and then decreased. High-throughput 16S rRNA gene pyrosequencing analysis indicated the accumulation of Actinobacteria, Chlorobaculum of Chlorobi and Proteiniphilum of Bacteroidetes, which might be responsible for the function of the proposed system. This study provides a step forward for the remediation of V(V) from polluted groundwater, by employing a promising biotechnology.

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1. Introduction

Vanadium is a widespread element in the environment due to its extensive application in modern industry. Therefore, its environmental impact has become an urgent issue.^1–3 The toxicity of vanadium increases with an increase in its valence state, with V(V) being the most toxic form,^4–6 while V(IV) is less toxic and insoluble at neutral pH.⁷ Consequently, the reduction of V(V) to V(IV) is considered to be effective for the remediation of V(V) contaminated groundwater, as the generated V(IV) can precipitate spontaneously and be removed through filtration.⁸ Physical and chemical methods are frequently employed, but their cost-effectiveness and secondary pollution are frequently questionable.^9,10

Recently, anaerobic microbial V(V) reduction has been recognized as a promising strategy for V(V) pollution remediation, as it is cost-effective and can be used for in situ remediation.¹¹ This technology has gained considerable interest and has included numerous pure strains such as Geobacter metallireducens, Shewanella oneidensis and Pseudomonas.^12–14 Mixed cultures with higher efficiencies compared to pure strains are readily available for actual remediation applications.^15–17 In the process of microbial metabolism, organic carbon sources have an important influence, especially for mixed cultures.^18,19 Regarding microbiological V(V) reduction, limited dissolved organic carbon sources have been employed.⁹ The metabolic effect of different carbon sources on V(V) reduction, as well as the accumulation of microbial communities, should be comparatively studied.

In the present study, the influence of five kinds of dissolved organic carbon sources (acetate, citrate, glucose, lactate and soluble starch) on microbial V(V) reduction was investigated. Operating factors and the microbes involved were examined with an optimum organic carbon source as well. The results were favorable for actual applications of bioremediation of V(V) in contaminated environments.

2. Methods and materials

2.1 Inoculated cultures and solutions

Ten 250 mL jars with silica gel stoppers acted as bioreactors. Each bioreactor was filled with 50 mL of anaerobic sludge collected from an up-flow anaerobic sludge blanket (UASB) reactor for treating high strength wastewater (Beijing YanJing Brewery CO., LTD, China) and 200 mL simulated groundwater containing the following components per liter: NH₄Cl (0.1557 g), CaCl₂ (0.2464 g), MgCl₂ (1.0572 g), NaCl (0.4459 g), KCl (0.0283 g), NaHCO₃ (0.8082 g) and KH₂PO₄ (0.0299 g). V(V) was added in the form of NaVO₃·2H₂O. The bioreactors were divided into five equal groups and were operated with acetate, glucose, citrate, lactate and soluble starch as organic carbon sources, respectively, with the same chemical oxygen demand (COD) concentration (800 mg L⁻¹ unless otherwise stated).

2.2 Experimental procedure

All bioreactors were filled with fresh groundwater containing a V(V) concentration of 75 mg L⁻¹, as well as the different dissolved organic carbon sources (800 mg L⁻¹) mentioned above, every 3 days for microbial domestication. This lasted 3 months for successful start-up before beginning the formal experiments. After that, microbial V(V) reduction with different dissolved organic carbon sources was evaluated in a 12 h fed-batch operation, as most V(V) was reduced in the bioreactors within that time. The corresponding reduced products were monitored as well. After that, influencing factors such as initial V(V) concentration (50 mg L⁻¹, 75 mg L⁻¹, 150 mg L⁻¹, 300 mg L⁻¹), initial COD concentration (400 mg L⁻¹, 800 mg L⁻¹, 1200 mg L⁻¹, 1600 mg L⁻¹), pH (6, 7, 8, 9) and conductivity (8 mS cm⁻¹, 10 mS cm⁻¹, 14 mS cm⁻¹, 16 mS cm⁻¹) were examined separately with the selected optimum substrate. The pH was adjusted by using HCl (1 mM) and NaOH (1 mM). The conductivity was adjusted by adding different doses of NaCl. The richness, diversity and taxonomy of the involved microbes with the optimum substrate were analyzed using high throughput sequencing after another three months of accumulation. All the experiments were conducted at room temperature (22 ± 2 °C). The two bioreactors in each group operated under identical conditions during the whole experiment. For each condition, trials were carried out in triplicate and the average results from the two bioreactors in the same group were reported.

2.3 Analytical methods and microbiological analysis

Spectrophotometric methods were chosen to measure the residual V(V) and generated V(IV).^20,21 Total vanadium was determined using ICP-MS (Thermo Fisher X series, Germany). COD was measured using a fast airtight catalytic decomposition method. pH and conductivity were measured using a pH-201 meter (Hanna, Italy).

Bacteria in the bioreactor containing the optimum substrate as well as in the inoculated sludge were collected by ultrasonication. Their total genomic DNA was extracted using a FastDNA® SPIN Kit for Soil (Qiagen, CA, the USA), according to the manufacturer's instructions. Then the above DNA was pooled and amplified using PCR (GeneAmp® 9700, ABI, the USA). After purification and quantification, a mixture of amplicons was taken for high-throughput 16S rRNA gene pyrosequencing on a MiSeq (Illumina, the USA). The sequences reported in the present study were submitted to the NCBI Sequence Read Archive with the study accession number of SRP056406.

3. Results and discussion

3.1 Microbial V(V) reduction with different dissolved organic carbon sources

With a V(V) concentration of 75 mg L⁻¹ and a COD of 800 mg L⁻¹, a gradual removal of V(V) was observed during the 12 h operation in all reactors, demonstrating that the mixed culture performed well and the selected organic carbon sources could support anaerobic microbial V(V) reduction successfully (Fig. 1). At the end of the 12 h operation, the V(V) removal efficiency reached between 65% and 76%, exhibiting an advantage to pure cultures and in situ V(V) reduction by indigenous microbes in groundwater.^22,23 During the microbial process in the bioreactors, V(V) acted as an electron acceptor and organics played the roles of electron donor and carbon source, thus reducing V(V).²⁴ Microbial V(V) reduction occurred in one of two ways: V(V) could be respired (via electron transfer) or it was reduced by the bacteria for detoxification purposes, as a result of vanadium binding to reductases of other electron acceptors, without evidence of respiration.²² Both of these two processes occurred due to the function of specific microbes, as mixed cultures were employed in the present study. Additionally, a green precipitate concomitantly appeared, its main component being the mineral sincosite, in line with our previous study.¹⁷ This implied that V(V) pollution can be alleviated through microbial reduction.¹⁷

Fig. 1 Time histories of V(V) concentrations in the bioreactors with five kinds of carbon sources during the 12 h operation.

Another observation in this work was that the performance of the bioreactors varied due to the different characteristics of the organics, as shown in Fig. 1. This finding is in agreement with previous studies.^25,26 V(V) removal efficiencies generally decreased with an increase in the molecular weight of the carbon substrate. In addition, organic acids supported higher V(V) removal than alcohols (Fig. 1). The microbes absorbed simpler organic compounds that could be oxidized more directly, resulting in outstanding performance.²⁷ Acetate has been observed to be a non-fermentative substrate, as well as a major fermentation product along with small molecule organic acids such as citrate and lactate, when a fermentative substrate such as glucose and soluble starch are employed during anaerobic fermentation processes.²⁸ Furthermore, the anaerobic fermentation process competes with the microbial V(V) reduction process with regards to electron capture, especially when fermentative substrates such as glucose and soluble starch are employed.¹⁷ As acetate has been reported to be a major organic electron donor that supports anaerobic respiration in subsurface environments,^9,22 it was chosen as an optimum carbon source in the present study and employed in the following experiments.

3.2 Influences of operating factors using an optimum carbon source

Four gradients of initial V(V) concentration were examined, with an initial COD concentration of 800 mg L⁻¹, pH of 7.0 and conductivity of 8 mS cm⁻¹. It can be seen from Fig. 2a that most V(V) was removed gradually within the 12 h operating period. With an increase in the initial concentration, the removal amount of total V(V) increased accordingly, however, the removal efficiency decreased. Microbial activity was inhibited due to a higher initial V(V) concentration, thus lowering the V(V) removal efficiency, as bacterial species have been shown to be tolerant to V(V) in the range of 110 mg L⁻¹ to 230 mg L⁻¹.²⁹ Our results are consistent with this finding, as a significant decrease in the V(V) removal efficiency was observed when the initial V(V) concentration was increased to 300 mg L⁻¹.

Fig. 2 Study investigating the factors affecting V(V) reduction in the bioreactors containing acetate: (a) initial V(V) concentration; (b) initial COD concentration; (c) pH and (d) conductivity.

As the activity of dissimilatory metal reduction bacteria is affected by the amount of electron donors and carbon sources, experiments employing different initial COD concentrations were conducted, with an initial V(V) concentration of 75 mg L⁻¹, pH of 7.0 and conductivity of 8 mS cm⁻¹. It can be seen from Fig. 2b that an appropriate increase in COD resulted in the improvement of V(V) reduction, but the efficiency decreased upon further increase in the initial COD concentration. As previously reported, approximately 500 mg L⁻¹ COD is required for microbes to reduce 75 mg L⁻¹ V(V).¹² When the concentration of the initial COD concentration is lower than 400 mg L⁻¹, there would be not enough electron donors and carbon sources to support microbe growth as well as V(V) reduction. When the initial COD concentration increases substantially, methanogenesis would compete with the dissimilatory metal reduction process at a higher initial COD concentration, as methanogenus can also employ non-fermentative acetate as an electron donor and a carbon source.⁹

Fig. 2c illustrates that the V(V) removal varied with varying pH, with an initial V(V) concentration of 75 mg L⁻¹, initial COD concentration of 800 mg L⁻¹, and conductivity of 8 mS cm⁻¹. The microbes could survive under all the tested pH values, and showed gradual V(V) removal, indicating that this bioremediation method could function over a relatively wide pH range. The removal efficiency was much higher under alkalescent conditions than under acidic or neutral ones. According to Bell et al.,³⁰ pH effects play an important role in the toxicity of vanadium salts in the nutrient broth. Our findings confirm that pH changes can affect the tolerance limits of test organisms to V(V) in mixed liquor.³¹ When the pH is high, the solubility of some metals decrease, while at the low pH, dissolved V(V) is released in aqueous solution³² where it can express its toxicity.

The effects of conductivity on V(V) reduction were also examined, using an initial V(V) concentration of 75 mg L⁻¹, initial COD concentration of 800 mg L⁻¹ and pH of 7.0. As the conductivity increased, the removal of V(V) first increased and then decreased (Fig. 2d). At lower conductivity levels, an increasing conductivity can promote contacts between the microbes and V(V). However, the microbial activity was inhibited due to the higher salt concentrations, thus the V(V) reduction efficiency declined.³³

3.3 Identification of the involved microbes

21 635 and 10 574 high-quality reads with an average length of 395 bp were recorded using high-throughput sequencing for the inocula and microbes in the bioreactor with acetate, consistent with other high-throughput studies.³⁴ Rarefaction curves, which were drawn at a 3% distance, also exhibited a change in the species richness of these two samples, due to three months' domestication using particular substrates (V(V) and acetate) in bioreactors (Fig. 3). The Shannon diversity index not only provides the species richness, but also reveals how the abundance of each species is distributed among all the species in the community.³⁴ This value decreased from 4.0 for the inoculated sludge to 1.4 for the bioreactor, which further proved that some microbes could not survive with V(V) present.

Fig. 3 Rarefaction curves based on pyrosequencing of inoculated sludge and bacterial communities in the bioreactors containing acetate. The OTUs were defined by 3% distances.

There were 27 genotypes of phylum discovered in the inoculated sludge, while only 10 genotypes of phylum were discovered in the bioreactor containing sodium acetate, indicating a significant change compared with the inocula (Fig. 4). During the whole operation, a large number of genotypes disappeared, while Actinobacteria, Chlorobi and Firmicutes increased significantly. Due to the changes in the living environment, structures of bacteria communities evolved.

Fig. 4 Bacterial community compositions at phylum level revealed by pyrosequencing of inoculated sludge and bacterial communities in the bioreactors containing acetate.

A taxonomy analysis of phylum, class and genus levels was performed to further study the microbial communities as well as their functions (Table 1). Some critical species responsible for V(V) reduction with energy from the oxidation of organic compounds under anaerobic environment were discovered. Species related to the dissimilatory reduction of metals that could also function in the reduction of V(V) were found, for which there have been very few direct reports published previously. For example, Actinobacteria, which has been shown to be able to cope with the presence of V(V)³⁵ and has been used to remediate soil co-contaminated with Cr(VI), was greatly accumulated.³⁶ The enriched Lactococcus of Firmicutes in the bioreactors has been reported to be able to reduce and precipitate silver in its metallic form.³⁷ These mentioned species might be conducive to V(V) reduction, when accompanied by other microbes.

Table 1 The percentages of sequences identified for different phylogenies in the bioreactors containing acetate

Phylum	Class	Genus	Bioreactor (%)	Phylum	Class	Genus	Bioreactor (%)
Acidobacteria	Acidobacteria	Holophaga	0.09	Firmicutes	Erysipelotrichia	Uncultured	0.07
		Norank	0.01		Negativicutes	Acidaminococcus	0.02
Actinobacteria	Actinobacteria	Bifidobacterium	0.02			Norank	0.77
		Norank	0.36			Uncultured	0.17
		Propionicicella	0.25	Lentisphaerae	Lentisphaeria	Victivallis	0.05
		Uncultured	63.2	Proteobacteria	Alphaproteobacteria	Bauldia	0.05
Armatimonadetes	Norank	Norank	0.01			Hyphomicrobium	0.01
Bacteroidetes	Bacteroidia	Macellibacteroides	0.13			Methylocystis	0.01
		Norank	0.03			Pleomorphomonas	1.31
		Paludibacter	0.59		Betaproteobacteria	Ferribacterium	0.01
		Petrimonas	0.06			Ideonella	0.01
		Proteiniphilum	1.59		Deltaproteobacteria	Desulfobulbus	0.68
		VadinBC27_wastewater-sludge_group	0.05			Desulfovibrio	0.05
	Sphingobacteriia	Norank	0.18			Norank	0.01
	WCHB1-32	Norank	0.03			Smithella	0.01
Chlorobi	Chlorobia	Chlorobaculum	21.85			Syntrophorhabdus	0.01
		Chlorobium	0.02			Uncultured	0.01
	Ignavibacteria	Norank	0.13		Epsilonproteobacteria	Sulfurospirillum	0.04
Chloroflexi	Anaerolineae	Leptolinea	0.01		Gammaproteobacteria	Enterobacter	0.14
Firmicutes	Bacilli	Lactococcus	0.23			Norank	0.04
	Clostridia	Acetobacterium	0.07			Pseudomonas	0.02
		Clostridium_sensu_stricto_1	0.05			Tolumonas	0.01
		Clostridium_sensu_stricto_5	0.05			Uncultured	0.01
		Eubacterium	0.25	Spirochaetae	Spirochaetes	Spirochaeta	0.71
		Incertae_Sedis	0.09			Uncultured	3.91
		Intestinimonas	0.01	Others			2.51

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There were also lots of fermentative microorganisms found in the bioreactors containing acetate, accompanied by metal reducing microbes. Proteiniphilum of Bacteroidetes, that was detected in the bioreactors, has been reported to be a member of the family of fermentative micro-organisms capable of producing acetate and hydrogen.³⁸ Paludibacter of Bacteroidetes, a type of fermentative bacteria that can ferment complex organics into the products of acetic, butyric and lactic acids and CO₂/H₂, was also found.³⁹ Spirochaeta of Spirochaetae, which has the ability to ferment carbohydrates into simple organic acids, also appeared.⁴⁰ Although these fermentative microorganisms could not reduce V(V) directly, they could survive with microbial metabolites as well as dead bacteria and interacted with metal reducing species to facilitate V(V) reduction.

As the inocula were collected from plant treating industry wastewater containing sulfate, sulfur related microbes were also detected in the bioreactor. Chlorobaculum of Chlorobi, a green sulfur bacterium, was greatly enriched.⁴¹ Desulfobulbus of Proteobacteria, which can grow with Fe(III) as an electron acceptor, was also found.⁴² These sulfur related microbes could function well with regards to electron transfer, which facilitated V(V) reduction through extracellular processes.

4. Conclusions

Acetate supported the highest V(V) removal efficiency of 75.6% during a 12 h operation in the bioreactors, compared with lactate, glucose, citrate and soluble starch. Operating factors, such as initial V(V), COD concentration, conductivity and pH, affected the performance of the bioreactors with regards to V(V) reduction. High-throughput 16S rRNA gene pyrosequencing analysis indicated the accumulation of Actinobacteria, Chlorobaculum of Chlorobi and Proteiniphilum of Bacteroidetes. They reacted together to realize effective V(V) reduction.

Acknowledgements

This research work was supported by the National Natural Science Foundation of China (NSFC) (No. 41440025, 21307117), the Research Fund for the Doctoral Program of Higher Education of China (No. 20120022120005), the Beijing Excellent Talent Training Project (No. 2013D009015000003), the Beijing Higher Education Young Elite Teacher Project (No. YETP0657) and the Fundamental Research Funds for the Central Universities (No. 2652015300, 2652015326).

References

1. J. X. Li, B. G. Zhang, Q. N. Song and A. G. L. Borthwick, RSC Adv., 2016, 6, 32940–32946.
2. L. Tian, J. Yang, C. Alewell and J. H. Huang, Chemosphere, 2014, 111, 89–95.
3. B. G. Zhang, C. X. Tian, Y. Liu, L. T. Hao, Y. Liu, C. P. Feng, Y. Liu and Z. Wang, Bioresour. Technol., 2015, 179, 91–97.
4. D. C. Crans and I. Boukhobza, J. Am. Chem. Soc., 1998, 120, 8069–8078.
5. P. Rasoulnia and S. M. Mousavi, RSC Adv., 2016, 6, 9139–9151.
6. H. Wang and Z. J. Ren, Water Res., 2014, 66, 219–232.
7. M. A. Larsson, M. D'Amato, F. Cubadda, A. Raggi, I. Öborn, D. B. Kleja and J. P. Gustafsson, Geoderma, 2015, 259–260, 271–278.
8. I. Ortiz-Bernad, R. T. Anderson, H. A. Vrionis and D. R. Lovley, Appl. Environ. Microbiol., 2004, 70, 3091–3095.
9. B. A. Reul, S. S. Amin, J. P. Buchet, L. N. Ongemba, D. C. Crans and S. M. Brichard, Br. J. Pharmacol., 1999, 126, 467–477.
10. L. T. Hao, B. G. Zhang, C. X. Tian, Y. Liu, C. H. Shi, M. Cheng and C. P. Feng, J. Power Sources, 2015, 287, 43–49.
11. Y. Li, G. K. C. Low, J. A. Scott and R. Amal, J. Hazard. Mater., 2007, 142, 153–159.
12. W. Carpentier, K. Sandra, D. I. Smet, A. Brige, D. L. Smet and J. Van Beeumen, Appl. Environ. Microbiol., 2003, 69, 3636–3639.
13. W. Carpentier, D. L. Smet, J. Van Beeumen and A. Brige, J. Bacteriol., 2005, 187, 3293–3301.
14. N. N. Lyalkova and N. A. Yurkova, Geomicrobiol. J., 1992, 10, 15–26.
15. S. O. Rastegar, S. M. Mousavi and S. A. Shojaosadati, RSC Adv., 2015, 5, 41088–41097.
16. B. G. Zhang, L. T. Hao, C. Tian, S. H. Yuan, C. P. Feng, J. R. Ni and A. G. L. Borthwick, Bioresour. Technol., 2015, 192, 410–417.
17. C. Y. Lai, L. L. Wen, Y. Zhang, S. S. Luo, Q. Y. Wang, Y. H. Luo, R. Chen, X. Yang, B. E. Rittmann and H. P. Zhao, Water Res., 2016, 88, 467–474.
18. N. Gartzia-Bengoetxea, E. Kandeler, I. M. de Arano and A. Arias-González, Appl. Soil Ecol., 2016, 100, 57–64.
19. S. Tripathy, P. Bhattacharyya, R. Mohapatra, A. Som and D. Chowdhury, Ecol. Eng., 2014, 70, 25–34.
20. A. Safavi, H. Abdollahi, F. Sedaghatpour and S. Zeinali, Anal. Chim. Acta, 2000, 409, 275–282.
21. A. A. Ensafi, M. K. Amini and M. Mazloum-Ardakani, Anal. Lett., 1999, 32, 1927–1937.
22. A. P. Yelton, K. H. Williams, J. Fournelle, K. C. Wrighton, K. M. Handley and J. F. Banfield, Environ. Sci. Technol., 2013, 47, 6500–6509.
23. J. van Marwijk, D. J. Opperman, L. A. Piater and E. van Heerden, Biotechnol. Lett., 2010, 31, 845–849.
24. J. Zhang, H. L. Dong, L. D. Zhao, R. McCarrick and A. Agrawal, Chem. Geol., 2014, 370, 29–39.
25. M. Mahmoud, P. Parameswaran, C. I. Torres and B. E. Rittmann, Bioresour. Technol., 2014, 151, 151–158.
26. L. T. Hao, B. G. Zhang, M. Cheng and C. P. Feng, Bioresour. Technol., 2016, 201, 105–110.
27. C. I. Torres, A. K. Marcus and B. E. Rittmann, Appl. Microbiol. Biotechnol., 2007, 77, 689–697.
28. S. Macfarlane and G. T. Macfarlane, Proc. Nutr. Soc., 2003, 62, 67–72.
29. I. Kamika and M. Momba, Water, Air, Soil Pollut., 2012, 223, 2525–2539.
30. J. M. Bell, J. C. Philp, M. S. Kuyukina, I. B. Ivshina, S. A. Dunbar, C. J. Cunningham and P. Anderson, J. Microbiol. Methods, 2004, 58, 87–100.
31. S. Freguia, K. Rabaey, Z. Yuan and J. Keller, Environ. Sci. Technol., 2008, 42, 7937–7943.
32. S. M. Shaheen, J. Rinklebe, T. Frohne, J. R. White and R. D. DeLaune, Chemosphere, 2015, 1–9.
33. B. G. Zhang, S. G. Zhou, H. Z. Zhao, C. H. Shi, L. C. Kong, J. J. Sun, Y. Yang and J. R. Ni, Bioprocess Biosyst. Eng., 2010, 33, 187–194.
34. L. Lu, D. Xing and N. Ren, Water Res., 2012, 46, 2425–2434.
35. R. Duran, A. Bielen, T. Paradžik, E. Pustijanac, C. Cagnon, B. Hamer and D. Vujaklija, Environ. Sci. Pollut. Res., 2015, 42, 1–15.
36. M. A. Polti, J. D. Aparicio, C. S. Benimeli and M. J. Amoroso, Int. Biodeterior. Biodegrad., 2014, 88, 48–55.
37. L. Sintubin, W. De Windt, J. Dick, J. Mast, D. van der Ha, W. Verstraete and N. Boon, Appl. Microbiol. Biotechnol., 2009, 84, 741–749.
38. J. F. Miceli III, P. Parameswaran, D. W. Kang, R. Krajmalnik-Brown and C. I. Torres, Environ. Sci. Technol., 2012, 46, 10349–10355.
39. J. X. Zhang, Y. B. Zhang, X. Quan, S. Chen and S. Afzal, Bioresour. Technol., 2013, 136, 273–280.
40. Y. Sun, Z. Jiane, L. T. Cui, D. Qian and D. Yang, J. Gen. Appl. Microbiol., 2010, 56, 19–29.
41. G. He, D. M. Niedzwiedzki, G. S. Orf, H. Zhang and R. E. Blankenship, J. Phys. Chem. B, 2015, 119, 8321–8329.
42. D. E. Holmes, D. R. Bond and D. R. Lovley, Appl. Environ. Microbiol., 2004, 70, 1234–1237.

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