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Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Simultaneous removal of perchlorate and energetic compounds in munitions wastewater by zero-valent iron and perchlorate-respiring bacteria a
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Se Chang Ahn , Brian Hubbard , Daniel K. Cha & Byung J. Kim
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Department of Civil and Environmental Engineering , University of Delaware , Newark , Delaware , USA b
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U.S. Army Engineering Research and Development Center , Champaign , Illinois , USA Published online: 10 Jan 2014.
To cite this article: Se Chang Ahn , Brian Hubbard , Daniel K. Cha & Byung J. Kim (2014) Simultaneous removal of perchlorate and energetic compounds in munitions wastewater by zero-valent iron and perchlorate-respiring bacteria, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:5, 575-583, DOI: 10.1080/10934529.2014.859455 To link to this article: http://dx.doi.org/10.1080/10934529.2014.859455
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Journal of Environmental Science and Health, Part A (2014) 49, 575–583 C Taylor & Francis Group, LLC Copyright ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2014.859455
Simultaneous removal of perchlorate and energetic compounds in munitions wastewater by zero-valent iron and perchlorate-respiring bacteria SE CHANG AHN1, BRIAN HUBBARD1, DANIEL K. CHA1 and BYUNG J. KIM2 1
Department of Civil and Environmental Engineering, University of Delaware, Newark, Delaware, USA U.S. Army Engineering Research and Development Center, Champaign, Illinois, USA
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Ammonium perchlorate is one of the main constituents in Army’s insensitive melt-pour explosive, PAX-21 in addition to RDX and 2,4-dinitroanisole (DNAN). The objective of this study is to develop an innovative treatment process to remove both perchlorate and energetic compounds simultaneously from PAX-21 production wastewater. It was hypothesized that the pretreatment of PAX21 wastewater with zero-valent iron (ZVI) would convert energetic compounds to products that are more amenable for biological oxidation and that these products serve as electron donors for perchlorate-reducing bacteria. Results of batch ZVI reduction experiments showed that DNAN was completely reduced to 2,4-diaminoanisole and RDX was completely reduced to formaldehyde. Anaerobic batch biodegradation experiments showed that perchlorate (30 mg L−1) in ZVI-treated PAX-21 wastewater was decreased to an undetectable level after 5 days. Batch biodegradation experiments also confirmed that formaldehyde in ZVI-treated wastewater was the primary electron donor for perchlorate-respiring bacteria. The integrated iron-anaerobic bioreactor system was effective in completely removing energetic compounds and perchlorate from the PAX-21 wastewater without adding an exogenous electron donor. This study demonstrated that ZVI pretreatment not only removed energetic compounds, but also transformed energetic compounds to products that can serve as the source of electrons for perchlorate-respiring bacteria. Keywords: Munitions wastewater, zero-valent iron, perchlorate, RDX, perchlorate-respiring bacteria.
Introduction US Army and Department of Defense (DoD) facilities generate ammunition wastewater containing a mixture of energetic compounds such as perchlorate (ClO4 −), nitroaromatic and nitramine compounds from munitions manufacturing and demilitarization processes. For example, ammonium perchlorate, hexahydro-1,3,5-trinitro1,3,5-triazine (RDX), and 2,4-dinitroanisole (DNAN) are the major constituents in the US Army’s new main charge melt-pour energetic, PAX-21. Perchlorate is known to have adverse effects on human health by interfering with the uptake of iodide into the thyroid gland. Iodide is an essential component for making thyroid hormones.[1,2] RDX is known to be toxic to various organisms including humans.[3,4] Due to its toxicity and possible carcinogenicity, the US EPA classifies RDX as a possible human Address correspondence to Daniel K. Cha, Department of Civil and Environmental Engineering, University of Delaware, Newark, DE 19716, USA; E-mail: [email protected] Received July 17, 2013. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lesa.
carcinogen (US EPA Class C). Presently, most Army ammunition plants use granular activated carbon (GAC) adsorption and alkaline hydrolysis to separate and treat energetic compounds in wastewater from munitions manufacturing and demilitarization processes. GAC processes are not only expensive but generate explosive-laden spent carbon, which needs to be treated or disposed of properly to avoid secondary contamination problems. This additional treatment further increases the overall cost of wastewater treatment. Aqueous perchlorate is both chemically stable in natural water and extremely soluble and mobile;[5] as a result, traditional wastewater treatment techniques that are commonly used for solvents and other organic pollutants are not effective for the removal of perchlorate from contaminated water.[6,7] Biological perchlorate removal has been considered as a highly effective and economically attractive option for the treatment of perchlorate-laden water.[8,9] Under anoxic conditions, perchlorate-respiring bacteria (PRB) use perchlorate as an electron acceptor for cellular respiration.[10] PRBs are ubiquitous in natural environment such as soils, sediments, surface water, and groundwater aquifers and they can grow by coupling oxidation of organic/ inorganic electron donors with the reduction of perchlorate
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576 under anaerobic conditions. Various PRB species have been isolated and many researchers reported that Dechloromonas and Azospira (formerly Dechlorosoma) are the dominant species in the environment.[6,10,11] Many different electron donors, including ethanol, fatty acids (acetate, lactate, propionate, and citrate) and vegetable oils, have been shown to support the growth of PRB.[9] Hydrogen gas has been shown to support autotrophic perchlorate reduction.[12–15] With the supply of hydrogen gas, 30–39% perchlorate reduction was achieved in an autotrophic bioreactor enriched with the strains of Dechloromonas sp.[13] Microbial reduction of perchlorate in the presence of zero-valent iron (ZVI) was reported and hydrogen gas released from iron corrosion was utilized by PRB as the primary source of electrons for perchlorate reduction.[16–18] Son et al.[16] reported that a column bioreactor containing iron granules and anaerobic mixed cultures completely removed 1000 µg L−1 of perchlorate at hydraulic residence time of 12 h. They suggested that the use of zero-valent iron granules can eliminate the need to continuously supply expensive organic electron donors or potentially dangerous hydrogen gas. Microbial treatment of perchlorate in munitions wastewater may be considered by coupling energetic compounds as electron donor and perchlorate as electron acceptor. Our preliminary experiments, however, showed that biological oxidation of major constituents in munitions wastewater was inefficient because energetic compounds have electron-withdrawing nitro constituents. Direct biooxidation of these nitroaromatic and nitramine compounds is
Fig. 1. Schematic diagram of ZVI and biological combined system.
Ahn et al. difficult due to the presence of electron-withdrawing nitro constituents that inhibit electrophilic attack by oxygenases enzymes.[19] Potential application of iron granules for enhancing aerobic biodegradation of highly oxidized aromatic compounds has recently been demonstrated.[20–22] Saxe et al.[21] showed that elemental iron pretreatment of azo dyecontaining wastewater can reductively transform the electron-withdrawing constituents on the azoaromatic compounds, which inhibit oxidative attack by microorganisms, and make them more amenable for biodegradation. Oh et al.[22] also showed that ZVI pretreatment transformed recalcitrant RDX to ring cleavage products that are more amenable to mineralization by aerobic bacteria. Ahn et al.[23] observed that ZVI pretreatment completely removed the primary toxic constituent in PAX-21 wastewater that inhibited the activity of perchlorate-reducing bacteria in the subsequent microbial process. The objective of this research was to develop an innovative treatment process to remove both perchlorate and highly oxidized energetic compounds from munitions manufacturing wastewater simultaneously. We hypothesized that ZVI reduction of energetic compounds will yield products that can serve as an electron donor for perchlorate reduction by PRB. The treatment system consists of (1) zero-valent iron process for the reduction of the electronwithdrawing groups on nitroaromatic and nitramine compounds and (2) an anaerobic biological treatment process for the microbial degradation of ZVI-treated explosive compounds using perchlorate as an electron
Removal of perchlorate in munitions wastewater by zero-valent iron acceptor (Fig. 1). With this combined system, the removal of both perchlorate and energetic compounds was achieved without adding any exogenous electron donor.
Materials and methods
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Microorganisms Activated sludge cultures obtained from the Wilmington wastewater treatment plant (Wilmington, DE, USA) were used as seed cultures for batch biodegradation studies. The culture medium contained 1.39 g L−1 Na2 HPO4 , 0.85 g L−1 KH2 PO4 , 0.1 g L−1 (NH4 )2 SO4 , 0.2 g L−1 MgSO4 ·7H2 O, 1 mL trace mineral solution, and 1 mL Ca-Fe solution (adapted from Nerenberg et al.[24]). The Ca-Fe solution contained 1 g L−1 CaCl2 ·2H2 O, 1 g L−1 FeSO4 ·7H2 O. The trace mineral solution contained 100 mg L−1 ZnSO4 ·7H2 O, 30 mg L−1 MnCl2 ·4H2 O, 300 mg L−1 H3 BO3 , 200 mg L−1 CoCl2 ·6H2 O, 10 mg L−1 CuCl2 ·2H2 O, 10 mg L−1 NiCl2 ·6H2 O, 30 mg L−1 Na2 MoO4 ·2H2 O and 30 mg L−1 Na2 SeO3 . Chemicals Sodium perchlorate monohydrate (NaClO4 ·H2 O, ∼100%) was purchased from Fisher Scientific (Pittsburgh, PA, USA). The 2,4-Dinitroanisole (C7 H6 N2 O5 , DNAN, 98%), and 2-Methoxy-5-nitroanlinine (C7 H8 N2 O3 , 98%) were purchased from Sigma Chemical (St. Louis, MO, USA). 4-Methoxy-1,3-phenylenediamine sulfate hydrate (2,4Diaminoanisole sulfate hydrate, 99.5%) was purchased from Chem Service (West Chester, PA, USA) and 4Methoxy-3-nitroanlinine (C7 H8 N2 O3 , 97.8%) was purchased from ChemPacific (Baltimore, MD, USA). PAX21 wastewater obtained from Holston Army ammunition plant (Kingsport, TN, USA). Zero-valent iron granules used in this study was purchased from Peerless Metal Powders (Detroit, MI, USA) and was used after sieving with 10– 20 mesh. The specific surface area of Peerless iron granules was previously determined to be 1.67 m2 g−1.[25] Batch iron reduction experiments Batch iron reduction experiments were conducted using 35 mL bottles containing 30 mL of PAX-21 wastewater and 5 g of iron granules. The solution was deoxygenated by purging with N2 gas for 30 min prior to adding iron granules. After iron granules were added, the bottles were placed horizontally on a rotary platform shaker (Lab-line, Melrose Park, IL, USA) and continuously stirred at 150 rpm. At selected time intervals, about 1.5 mL of samples were taken from each bottle and passed through a 0.22-µm cellulose filter (Millipore, Billerica, MA, USA) for chemical analysis using a high performance liquid chromatography (HPLC).
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Batch biodegradation experiments To investigate whether iron-treated energetic compounds can serve a source of electrons for microbial reduction of perchlorate, batch biodegradation experiments were conducted using 250 mL amber bottles (liquid volume = 150 mL) incubated at room temperature under anaerobic conditions. Five sets of experimental bottles were prepared with the following treatments: (i) cells + PAX-21 wastewater (PAX-21 control), (ii) cells + 50 mg L−1 perchlorate (cell control), (iii) cells + 50 mg L−1 perchlorate + formaldehyde, (iv) cells + 50 mg L−1 perchlorate + 2,4-diaminoanisole (DAAN), and (v) cells + iron treated PAX-21 wastewater. Each bottle was seeded with an initial cell concentration of 500 mg L−1. All experimental reactors were prepared in an anaerobic glove box filled with N2 gas (Bell-Art Products, Pequannock, NJ, USA). All bottles were placed on a rotary platform shaker (Lab-line, Melrose Park, IL, USA) in a horizontal position and continuously stirred at 150 rpm. At different elapsed times, about 2 mL of samples were taken from each bottle and passed through a 0.22-µm cellulose filter (Millipore) for chemical analysis.
Operation of integrated iron-bioreactor system An integrated iron-bioreactor system consisted of a glass column (1.5 cm ID × 20 cm L, Ace Glass, Vineland, NJ, USA) packed with Peerless iron granules (porosity = 0.65) and an anaerobic bioreactor system. An upflow anaerobic sludge bioreactor system for this study consisted of an acrylic column (3.81 cm ID × 80 cm long) and a 500mL Erlenmeyer flask. The acrylic column was filled with perchlorate-reducing cultures established from an activated sludge sample obtained from the Wilmington wastewater treatment plant (Wilmington, DE, USA). Glass beads were placed at the bottom of the acrylic column to distribute the flow evenly across the column cross-section. The nutrient solution was pumped from the 500-mL reservoir (300 mL liquid volume) using a Masterflex peristaltic pump (ColeParmer, Vernon Hills, IL, USA) to the bottom of the column bioreactor and the column effluent was returned to the reservoir from the top by gravity. A flow rate of 150 mL min−1 was determined as the optimum upflow rate for maintaining a constant sludge blanket height,[26] which allowed the recirculation of clear supernatant back to the reservoir. The nutrient reservoir also served as the mixing chamber to combine the iron-treated PAX-21 stream from the iron column with nutrient solution and recirculation stream. The constituents of nutrient solution were: 1.4 g L−1 Na2 HPO4 , 0. 9 g L−1 KH2 PO4 , 0.1 g L−1 (NH4 )2 SO4 , 0.2 g L−1 MgSO4 ·7H2 O, 1 mL trace mineral solution, and 1 mL Ca-Fe solution (adapted from Nerenberg et al. [24]). The Ca-Fe solution contained 1 g L−1 CaCl2 ·2H2 O, 1 g L−1 FeSO4 ·7H2 O. The trace mineral solution contained 100 mg L−1 ZnSO4 ·7H2 O, 30 mg L−1 MnCl2 ·4H2 O, 300 mg L−1 H3 BO3 , 200 mg/L CoCl2 ·6H2 O,
578 10 mg L−1 CuCl2 ·2H2 O, 10 mg L−1 NiCl2 ·6H2 O, 30 mg L−1 Na2 MoO4 ·2H2 O and 30 mg L−1 Na2 SeO3 . A second peristaltic pump (Cole-Palmer, Chicago, IL, USA) was used to supply the ZVI-treated PAX-21 wastewater to the nutrient reservoir at a rate of 0.1 L h−1. The treated effluent from the integrated system was withdrawn from the top of the bioreactor column at the same flow rate of 0.1 L h−1. Prior to a continuous feeding operation, the bioreactor system was operated in a batch-feeding mode for 4 weeks to establish acclimated perchlorate-reducing cultures. A control system receiving untreated PAX-21 wastewater was operated in parallel.
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Analytical methods Perchlorate was analyzed using a Dionex ICS-1000 ion chromatograph (Dionex, Sunnyvale, CA, USA) equipped with an IonPAC AS16 2-mm column, a AG16 2-mm guard column, and ASRS Ultra 2-mm suppressor. NaOH solution (65 mM) was used as the eluent and the injection volume was 25 µL. DNAN, 2-Methoxy-5-nitroanlinine, and 4Methoxy-3-nitroanlinine, RDX, DAAN and formaldehyde were analyzed using a Dionex HPLC (Dionex) equipped with a Supelguard LC-18 guard column (length, 20 mm; inner diameter, 4.0 mm; Supelco, Bellefonte, PA, USA) and a Supelcosil LC-18 column (length, 250 mm; inner diameter, 4.6 mm; film thickness, 5 mm). The mixture of methanol and deionized water (50/50, v/v) was used as an eluent at a flow rate of 1.0 mL/min for measuring DNAN, 2Methoxy-5-nitroanlinine, 4-Methoxy-3-nitroanlinine, and RDX. The wavelength for the UV detector was 275 nm for 2-Methoxy-5-nitroanlinine and 254 nm for DNAN, 4-Methoxy-3-nitroanlinine, and RDX. For DAAN, phosphate buffer (20mM, pH 7)-acetonitrile (70/30, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The wavelength for the UV detector was 275 nm. Formaldehyde concentrations were determined after derivatization with Nash’s reagent (0.02 M of 2,4-pentanedione and 2 M of ammonium acetate, pH 6).[27] One milliliter of aqueous formaldehyde sample was derivatized with 1 mL of Nash’s reagent for 1 h at 60◦ C in a water bath (Precision, Winchester, VA, USA). After the sample was cooled to room temperature (20 ± 2◦ C), concentrations of formaldehyde derivative were determined using the Dionex HPLC [wavelength for the UV detector 410 nm, eluent the mixture of acetonitrile and deionized water (70/30, v/v)]. The injection volumes for all samples were 250 µL. Ammonium ion was analyzed with the Hach spectrophotometer using the salicylate method (Hach, Loveland, CO, USA).
Results and discussion Iron reduction experiments PAX-21 wastewater obtained from Holston Army ammunition plant contained 49.5 mg L−1 of RDX, 123.6 mg L−1
Ahn et al. of DNAN, and 30.2 mg L−1 of perchlorate. Fig. 2 shows the concentrations of DNAN, reduction intermediates (2Methoxy-5-nitroanlinine and 4-Methoxy-3-nitroanlinine), the end product (DAAN), and total carbon during the reduction of PAX-21 wastewater with zero-valent iron (ZVI). DNAN was rapidly and completely removed by iron granules. Approximately 60% of the initial DNAN disappeared within 10 min and complete removal was achieved after 60 min. Less than 30% of reduced DNAN was recovered as intermediates in the first 30 min of reaction time. Among the reduction intermediates measured, 2Methoxy-5-nitroaniline was the dominant intermediate, indicating that reduction of the ortho nitro group was the primary reaction pathway for DNAN reduction by ZVI. Fig. 2 also shows that 100% carbon recovery was achieved in 2 h. The lower carbon recovery during the first 60 min may be due to adsorption of DNAN and reduction intermediates to iron surface, and accumulation of intermediates that were not measured in this study. Oh et al.[28] also reported that more than 70% of DNT removal in the first 10 min of reaction time was attributed to adsorption by iron granules. RDX in PAX-21 wastewater was also completely removed by ZVI granules in 2 h (data not shown). The batch removal rate of RDX in PAX-21 by ZVI was similar to RDX reduction rates reported by Oh et al.[28] The major end products of RDX reduction by zero-valent iron were formaldehyde (15.8 mg L−1) and NH4 + (12.5 mg L−1). The batch reduction experiments showed that DNAN and RDX in PAX-21 wastewater can be readily transformed to DAAN and formaldehyde by ZVI granules.
Batch biodegradation experiments with ZVI-treated PAX-21 wastewater To examine whether ZVI-treated energetic compounds in PAX-21 can serve as electron donor sources for perchloraterespiring bacteria, a series of batch biodegradation tests were conducted with iron-treated PAX-21 wastewater. PAX-21 wastewater was initially passed through an ironpacked glass column (2.5 cm ID × 30 cm L) at a flow rate corresponding to a 15-min contact time. ZVI column treatment resulted in complete removal of DNAN and RDX from PAX-21 wastewater and the column effluent contained 87.8 mg L−1 of DAAN, 20.5 mg L−1 of HCHO and 30 mg L−1 of perchlorate. Fig. 3 shows the disappearance of perchlorate in PAX-21 wastewater and ZVI-treated PAX-21 wastewater in batch bioreactors. Perchlorate in the iron-treated PAX-21 wastewater decreased to an undetectable level after 5 days of incubation in anaerobic bioreactors, yet negligible perchlorate was removed from reactors containing untreated PAX-21 wastewater. Without iron pre-treatment, the energetic compounds in PAX-21 wastewater was not able to support microbial perchlorate reduction; however, after ZVI treatment, complete removal of perchlorate was obtained without adding
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external sources of electron donors. This result clearly demonstrated that ZVI pretreatment not only removed energetic compounds, but also transformed energetic compounds to products that can serve as the source of electrons for perchlorate-respiring bacteria. To further validate that the microbial reduction of perchlorate can be coupled with ZVI-treated energetic compounds in PAX-21 wastewater, batch bioreactors containing perchlorate were spiked with iron-reduction products of RDX (formaldehyde) and DNAN (DAAN). Fig. 4 shows the disappearance of perchlorate in batch reactors containing 40 mg L−1 of formaldehyde. Aqueous perchlorate (30 mg L−1) began to decrease immediately and complete removal of perchlorate was achieved in 5 days of incubation. On the other hand, negligible amount of perchlorate was removed in 6 days of incubation in the control reactor containing no formaldehyde. This result suggested that formaldehyde can serve as the electron donor for the reduction of perchlorate by perchlorate respiration bacteria. Figure 5 shows the effect of DAAN addition (100 mg L−1) on microbial reduction of perchlorate by the same perchlorate-respiring bacteria. In contrast to formaldehyde, the addition of 100 mg/L DAAN did not support the reduction of perchlorate as the disappearance of aqueous perchlorate was similar to the removal rate observed in cell control. Because batch degradation experiments were carried out with non-acclimated activated sludge as seed cultures, the inability of DAAN to support microbial perchlorate reduction during this study may be due to the lack
of acclimated microorganisms. It has been reported that the microbial oxidation of aminoaromatic compounds proceeds only with acclimated cultures that typically require months for establishment.[29,30] Pesce and Wunderlin [30] reported activated sludge microorganisms required a 4-month acclimation period in batch reactors for the treatment of 2,4-DAT- and 2,6DAT-containing wastewaters. Since DAAN was not able to support microbial perchlorate reduction by the cultures used in this study, the removal of perchlorate (30.2 mg L−1) in the PAX-21 solution was most likely coupled with formaldehyde (20.5 mg L−1) from the RDX reduction reaction. Formaldehyde concentration in the iron-treated PAX-21 was greater than the stoichiometric amounts needed for the complete reduction of perchlorate in the solution. Operation of integrated ZVI-bioreactor system To demonstrate the application of ZVI technology for simultaneous removal of perchlorate and energetic compounds, PAX-21 wastewater was continuously treated with the integrated ZVI-bioreactor system. The integrated system was initially supplemented with the daily addition of acetate (150 mg L−1 NaCH3 COO) and perchlorate (30 mg L−1) for about 4 weeks to establish stable perchloratereducing populations in the upflow sludge blanket reactor. After 4 weeks of culture enrichment, daily addition of acetate and perchlorate was stopped and ZVI-treated
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PAX-21 wastewater containing about 30 mg L−1 perchlorate was continuously fed to the nutrient reservoir at 0.1 L h−1. Figure 6 compares the effluent perchlorate concentrations from the ZVI integrated system and control unit over the 14-day experimental period. Perchlorate concentrations in the effluent of the integrated system were consistently below the detection limit (0.02 mg L−1) (Fig. 6), while the perchlorate concentrations in the effluent of the control system without ZVI treatment were greater than 20 mg L−1. This study demonstrated that ZVI column transformed the energetic compounds in PAX-21 wastewater to products that supported the growth of an anaerobic culture, which was capable of completely reducing aqueous perchlorate.
Conclusions Results of batch experiments showed that DNAN was completely reduced to 2,4-diaminoanisole (DAAN) and RDX was completely reduced to formaldehyde in the presence of cast iron granules within 2 h. Perchlorate (30 mg/L) in ZVI-treated PAX-21 wastewater was decreased to an undetectable level after 5 days of incubation in a batch bioreactor containing seed microorganisms obtained from a wastewater treatment plant. Without ZVI pre-treatment, the energetic compounds in PAX-21 wastewater was not able to support microbial perchlorate reduction. Batch biodegradation experiments with ZVI-reduction products of energetic compounds showed that formaldehyde can serve as an electron donor for perchloraterespiring bacteria. On the other hand, DNAN reduction product, DAAN, was not able to support the perchloratereducing activity by PRB. Anaerobic batch experiments demonstrated that complete reduction of perchlorate in iron-treated PAX-21 wastewater can be achieved without adding an exogenous electron donor. The integrated ZVIanaerobic bioreactor system was effective in removing perchlorate from PAX-21 wastewater without the addition of external electron donors. This study demonstrated that ZVI pretreatment not only removed energetic compounds, but also transformed the energetic compounds to products that can serve as the source of electrons for perchlorate-respiring bacteria. Use of zero-valent iron may eliminate the need to continually supply electron donors such as organic substrates to remove perchlorate.
References [1] Clark, J.J.J. Toxicology of perchlorate. In Perchlorate in the Environment; Urbanski, E.T., Ed.; Kluwer Academics/Plenum Publishers: New York, 2005; 15–29. [2] National Research Council. Health Implication of Perchlorate Ingestion; The National Academies Press: Washington, DC, 2006.
Ahn et al. [3] Urbanski, T. Chemistry and Technology of Explosives, Vol. 4; Pergamon: New York, 1985. [4] Yinon, J. Toxicity and Metabolism of Explosives; CRC Press: Boca Raton, FL, 1990. [5] Urbansky E.T.; Schock, M.R. Issues in managing the risks associated with perchlorate in drinking water. J. Environ. Manag. 1999, 56, 79–95. [6] Xu, J.; Song Y.; Min, B.; Steinberg, L.; Logan, B.E. Microbial degradation of perchlorate: principles and applications. Environ. Sci. Technol. 2003, 20, 405–422. [7] Council on Water Quality (Sacramento, CA). The history of perchlorate: evolving science, technologies and regulation 2004. Available at http://www.councilonwaterqaulity.org/science/ history.html (accessed Feb 2008). [8] US Environmental Protection Agency (US EPA). Perchlorate treatment technology update Author: Washington, DC, 2005. Available at http://www.epa.gov/tio/download/remed/ 542-r-05–015.pdf (accessed Feb 2008). [9] Hatzinger, P.B. Perchlorate biodegradation for water treatment. Environ. Sci. Technol. 2005, 39, 239A–247A. [10] Coates, J.D.; Achenbach, L.A. Microbial perchlorate reduction: rocket-fuelled metabolism. Nat. Rev. Microbiol. 2004, 2, 569– 580. [11] Coates, J.D.; Michaelidou, U.; Bruce, R.A.; O’Connor, S.M.; Crespi, J.N.; Achenbach, L.A. The ubiquity and diversity of dissimilatory (per)chlorate-reducing bacteria. Appl. Environ. Microbiol. 1999, 65, 5234–5241. [12] Miller J.P.; Logan, B.E. Sustained perchlorate degradation in an autotrophic, gas phase, packed-bed bioreactor. Environ. Sci. Technol. 2000, 34, 3018–3022. [13] Logan, B.E.; LaPoint, D. Treatment of perchlorate- and nitratecontaminated groundwater in an autotrophic, gas phase, packedbed bioreactor. Water Res. 2002, 36, 3647–3653. [14] Ahn, C.H.; Oh, H.; Ki, D.; Van Ginkel, S.W.; Rittmann, B.E.; Park, J. Bacterial biofilm-community selection during autohydrogenotrophic reduction of nitrate and perchlorate in ion-exchange brine. Appl. Microbiol. Biotechnol. 2009, 81, 1169–1170. [15] Sahu, A.K.; Conneely, T.; Nusslein, K.; Ergas, S.J. Hydrogenotrophic denitrification and perchlorate reduction in ion exchange brine using membrane biofilm reactors. Biotechnol. Bioeng. 2009, 10, 483–491. [16] Son, A.; Lee, J.; Chiu, P.C.; Kim, B.J.; Cha, D.K. Microbial reduction of perchlorate with zero-valent iron. Water Res. 2006, 40, 2027–2032. [17] Yu, X.; Amrhein, C.; Deshusses, M.A.; Matsumoto, M.R. Perchlorate reduction by autotrophic bacteria in the presence of zero-valent iron. Environ. Sci. Technol. 2006, 40, 1328–1334. [18] Son, A.; Schmidt, C.J.; Shin, H.; Cha, D.K. Microbial community analysis of perchlorate-reducing culturesgrowing on zero-valent iron. J. Hazard Mater. 2011, 185, 669–676. [19] VanderLoop, S.L.; Suidan, M.T.; Moteleb, M.A.; Maloney, S.W. Two-stage biotransformation of 2,4,6-trinitrotoluene. Bioremediation, 1995, 3(7), 225. [20] Perey, J.R.; Chiu, P.C.; Huang, C.P.; Cha, D.K. Zero-valent iron pretreatment for enhancing the biodegradability of azo dyes. Water Environ. Res. 2002, 74, 221–225. [21] Saxe, J.P.; Lubenow, B.L.; Chiu, P.C.; Huang, C.P.; Cha, D.K. Enhanced biodegradation of azo dyes using an integrated elemental iron-activated sludge system: I. Evaluation of system performance. Water Environ. Res. 2006, 78, 19–25. [22] Oh, S.Y.; Chiu, P.C.; Kim, B.J.; Cha, D.K. Zero-valent iron pretreatment for enhancing the biodegradability of RDX. Water Res. 2005, 39, 5027–5032. [23] Ahn, S.C.; Kim, B.J.; Cha, D.K.; Oh, S.Y. Detoxification of PAX-21 ammunitions wastewater by zero-valent iron for microbial reduction of perchlorate. J. Hazard. Mater. 2011, 192, 909–914.
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[24] Nerenberg, R.; Kawagoshi, Y.; Rittmann, B.E. Kinetics of a hydrogen-oxidizing, perchlorate-reducing bacterium, Water Res. 2006, 40, 3290–3296. [25] Oh, S.Y.; Chiu, P.C.; Kim, B.J.; Cha, D.K. Zero-valent iron treatment of RDX-containing and perchlorate-containing wastewaters from an ammunition-manufacturing plant at elevated temperatures. Water Sci. Technol. 2006, 54, 47–53. [26] Hubbard, B. Design and Operation of Novel Up-flow Bioreactor for Microbial Perchlorate Removal. Master’s thesis, University of Delaware, Newark, DE, 2008. [27] Oh, S.Y.; Cha, D.K.; Kim, B.J.; Chiu, P.C. Reductive transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine, octahydro-
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1,3,5,7-tetranitro-1,3,5,7-tetrazocine, and methylenedinitramine with elemental iron. Environ. Toxicol. Chem. 2005, 24, 2812– 2819. [28] Oh, S.Y.; Cha, D.K.; Chiu, P.C. Graphite-mediated reduction of 2,4-dinitrotoluene with elemental iron. Environ. Sci. Technol. 2002, 36, 2178–2184. [29] Freedman, D.L.; Shanley, R.S.; Scholze, R.J. Aerobic biodegradation of 2,4-dinitrotoluene, aminonitrotoluene isomers, and 2,4diamintoluene. J. Hazard. Mater. 1996, 49, 1–14. [30] Pesce, S.F.; Wunderlin, D.A. Biodegradation of 2.4- and 2,6diatninotoluene by acclimated bacteria. Water Res. 1997, 31, 1601– 1608.
Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Simultaneous removal of perchlorate and energetic compounds in munitions wastewater by zero-valent iron and perchlorate-respiring bacteria a
a
a
Se Chang Ahn , Brian Hubbard , Daniel K. Cha & Byung J. Kim
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Department of Civil and Environmental Engineering , University of Delaware , Newark , Delaware , USA b
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U.S. Army Engineering Research and Development Center , Champaign , Illinois , USA Published online: 10 Jan 2014.
To cite this article: Se Chang Ahn , Brian Hubbard , Daniel K. Cha & Byung J. Kim (2014) Simultaneous removal of perchlorate and energetic compounds in munitions wastewater by zero-valent iron and perchlorate-respiring bacteria, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:5, 575-583, DOI: 10.1080/10934529.2014.859455 To link to this article: http://dx.doi.org/10.1080/10934529.2014.859455
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Journal of Environmental Science and Health, Part A (2014) 49, 575–583 C Taylor & Francis Group, LLC Copyright ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2014.859455
Simultaneous removal of perchlorate and energetic compounds in munitions wastewater by zero-valent iron and perchlorate-respiring bacteria SE CHANG AHN1, BRIAN HUBBARD1, DANIEL K. CHA1 and BYUNG J. KIM2 1
Department of Civil and Environmental Engineering, University of Delaware, Newark, Delaware, USA U.S. Army Engineering Research and Development Center, Champaign, Illinois, USA
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Ammonium perchlorate is one of the main constituents in Army’s insensitive melt-pour explosive, PAX-21 in addition to RDX and 2,4-dinitroanisole (DNAN). The objective of this study is to develop an innovative treatment process to remove both perchlorate and energetic compounds simultaneously from PAX-21 production wastewater. It was hypothesized that the pretreatment of PAX21 wastewater with zero-valent iron (ZVI) would convert energetic compounds to products that are more amenable for biological oxidation and that these products serve as electron donors for perchlorate-reducing bacteria. Results of batch ZVI reduction experiments showed that DNAN was completely reduced to 2,4-diaminoanisole and RDX was completely reduced to formaldehyde. Anaerobic batch biodegradation experiments showed that perchlorate (30 mg L−1) in ZVI-treated PAX-21 wastewater was decreased to an undetectable level after 5 days. Batch biodegradation experiments also confirmed that formaldehyde in ZVI-treated wastewater was the primary electron donor for perchlorate-respiring bacteria. The integrated iron-anaerobic bioreactor system was effective in completely removing energetic compounds and perchlorate from the PAX-21 wastewater without adding an exogenous electron donor. This study demonstrated that ZVI pretreatment not only removed energetic compounds, but also transformed energetic compounds to products that can serve as the source of electrons for perchlorate-respiring bacteria. Keywords: Munitions wastewater, zero-valent iron, perchlorate, RDX, perchlorate-respiring bacteria.
Introduction US Army and Department of Defense (DoD) facilities generate ammunition wastewater containing a mixture of energetic compounds such as perchlorate (ClO4 −), nitroaromatic and nitramine compounds from munitions manufacturing and demilitarization processes. For example, ammonium perchlorate, hexahydro-1,3,5-trinitro1,3,5-triazine (RDX), and 2,4-dinitroanisole (DNAN) are the major constituents in the US Army’s new main charge melt-pour energetic, PAX-21. Perchlorate is known to have adverse effects on human health by interfering with the uptake of iodide into the thyroid gland. Iodide is an essential component for making thyroid hormones.[1,2] RDX is known to be toxic to various organisms including humans.[3,4] Due to its toxicity and possible carcinogenicity, the US EPA classifies RDX as a possible human Address correspondence to Daniel K. Cha, Department of Civil and Environmental Engineering, University of Delaware, Newark, DE 19716, USA; E-mail: [email protected] Received July 17, 2013. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lesa.
carcinogen (US EPA Class C). Presently, most Army ammunition plants use granular activated carbon (GAC) adsorption and alkaline hydrolysis to separate and treat energetic compounds in wastewater from munitions manufacturing and demilitarization processes. GAC processes are not only expensive but generate explosive-laden spent carbon, which needs to be treated or disposed of properly to avoid secondary contamination problems. This additional treatment further increases the overall cost of wastewater treatment. Aqueous perchlorate is both chemically stable in natural water and extremely soluble and mobile;[5] as a result, traditional wastewater treatment techniques that are commonly used for solvents and other organic pollutants are not effective for the removal of perchlorate from contaminated water.[6,7] Biological perchlorate removal has been considered as a highly effective and economically attractive option for the treatment of perchlorate-laden water.[8,9] Under anoxic conditions, perchlorate-respiring bacteria (PRB) use perchlorate as an electron acceptor for cellular respiration.[10] PRBs are ubiquitous in natural environment such as soils, sediments, surface water, and groundwater aquifers and they can grow by coupling oxidation of organic/ inorganic electron donors with the reduction of perchlorate
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576 under anaerobic conditions. Various PRB species have been isolated and many researchers reported that Dechloromonas and Azospira (formerly Dechlorosoma) are the dominant species in the environment.[6,10,11] Many different electron donors, including ethanol, fatty acids (acetate, lactate, propionate, and citrate) and vegetable oils, have been shown to support the growth of PRB.[9] Hydrogen gas has been shown to support autotrophic perchlorate reduction.[12–15] With the supply of hydrogen gas, 30–39% perchlorate reduction was achieved in an autotrophic bioreactor enriched with the strains of Dechloromonas sp.[13] Microbial reduction of perchlorate in the presence of zero-valent iron (ZVI) was reported and hydrogen gas released from iron corrosion was utilized by PRB as the primary source of electrons for perchlorate reduction.[16–18] Son et al.[16] reported that a column bioreactor containing iron granules and anaerobic mixed cultures completely removed 1000 µg L−1 of perchlorate at hydraulic residence time of 12 h. They suggested that the use of zero-valent iron granules can eliminate the need to continuously supply expensive organic electron donors or potentially dangerous hydrogen gas. Microbial treatment of perchlorate in munitions wastewater may be considered by coupling energetic compounds as electron donor and perchlorate as electron acceptor. Our preliminary experiments, however, showed that biological oxidation of major constituents in munitions wastewater was inefficient because energetic compounds have electron-withdrawing nitro constituents. Direct biooxidation of these nitroaromatic and nitramine compounds is
Fig. 1. Schematic diagram of ZVI and biological combined system.
Ahn et al. difficult due to the presence of electron-withdrawing nitro constituents that inhibit electrophilic attack by oxygenases enzymes.[19] Potential application of iron granules for enhancing aerobic biodegradation of highly oxidized aromatic compounds has recently been demonstrated.[20–22] Saxe et al.[21] showed that elemental iron pretreatment of azo dyecontaining wastewater can reductively transform the electron-withdrawing constituents on the azoaromatic compounds, which inhibit oxidative attack by microorganisms, and make them more amenable for biodegradation. Oh et al.[22] also showed that ZVI pretreatment transformed recalcitrant RDX to ring cleavage products that are more amenable to mineralization by aerobic bacteria. Ahn et al.[23] observed that ZVI pretreatment completely removed the primary toxic constituent in PAX-21 wastewater that inhibited the activity of perchlorate-reducing bacteria in the subsequent microbial process. The objective of this research was to develop an innovative treatment process to remove both perchlorate and highly oxidized energetic compounds from munitions manufacturing wastewater simultaneously. We hypothesized that ZVI reduction of energetic compounds will yield products that can serve as an electron donor for perchlorate reduction by PRB. The treatment system consists of (1) zero-valent iron process for the reduction of the electronwithdrawing groups on nitroaromatic and nitramine compounds and (2) an anaerobic biological treatment process for the microbial degradation of ZVI-treated explosive compounds using perchlorate as an electron
Removal of perchlorate in munitions wastewater by zero-valent iron acceptor (Fig. 1). With this combined system, the removal of both perchlorate and energetic compounds was achieved without adding any exogenous electron donor.
Materials and methods
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Microorganisms Activated sludge cultures obtained from the Wilmington wastewater treatment plant (Wilmington, DE, USA) were used as seed cultures for batch biodegradation studies. The culture medium contained 1.39 g L−1 Na2 HPO4 , 0.85 g L−1 KH2 PO4 , 0.1 g L−1 (NH4 )2 SO4 , 0.2 g L−1 MgSO4 ·7H2 O, 1 mL trace mineral solution, and 1 mL Ca-Fe solution (adapted from Nerenberg et al.[24]). The Ca-Fe solution contained 1 g L−1 CaCl2 ·2H2 O, 1 g L−1 FeSO4 ·7H2 O. The trace mineral solution contained 100 mg L−1 ZnSO4 ·7H2 O, 30 mg L−1 MnCl2 ·4H2 O, 300 mg L−1 H3 BO3 , 200 mg L−1 CoCl2 ·6H2 O, 10 mg L−1 CuCl2 ·2H2 O, 10 mg L−1 NiCl2 ·6H2 O, 30 mg L−1 Na2 MoO4 ·2H2 O and 30 mg L−1 Na2 SeO3 . Chemicals Sodium perchlorate monohydrate (NaClO4 ·H2 O, ∼100%) was purchased from Fisher Scientific (Pittsburgh, PA, USA). The 2,4-Dinitroanisole (C7 H6 N2 O5 , DNAN, 98%), and 2-Methoxy-5-nitroanlinine (C7 H8 N2 O3 , 98%) were purchased from Sigma Chemical (St. Louis, MO, USA). 4-Methoxy-1,3-phenylenediamine sulfate hydrate (2,4Diaminoanisole sulfate hydrate, 99.5%) was purchased from Chem Service (West Chester, PA, USA) and 4Methoxy-3-nitroanlinine (C7 H8 N2 O3 , 97.8%) was purchased from ChemPacific (Baltimore, MD, USA). PAX21 wastewater obtained from Holston Army ammunition plant (Kingsport, TN, USA). Zero-valent iron granules used in this study was purchased from Peerless Metal Powders (Detroit, MI, USA) and was used after sieving with 10– 20 mesh. The specific surface area of Peerless iron granules was previously determined to be 1.67 m2 g−1.[25] Batch iron reduction experiments Batch iron reduction experiments were conducted using 35 mL bottles containing 30 mL of PAX-21 wastewater and 5 g of iron granules. The solution was deoxygenated by purging with N2 gas for 30 min prior to adding iron granules. After iron granules were added, the bottles were placed horizontally on a rotary platform shaker (Lab-line, Melrose Park, IL, USA) and continuously stirred at 150 rpm. At selected time intervals, about 1.5 mL of samples were taken from each bottle and passed through a 0.22-µm cellulose filter (Millipore, Billerica, MA, USA) for chemical analysis using a high performance liquid chromatography (HPLC).
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Batch biodegradation experiments To investigate whether iron-treated energetic compounds can serve a source of electrons for microbial reduction of perchlorate, batch biodegradation experiments were conducted using 250 mL amber bottles (liquid volume = 150 mL) incubated at room temperature under anaerobic conditions. Five sets of experimental bottles were prepared with the following treatments: (i) cells + PAX-21 wastewater (PAX-21 control), (ii) cells + 50 mg L−1 perchlorate (cell control), (iii) cells + 50 mg L−1 perchlorate + formaldehyde, (iv) cells + 50 mg L−1 perchlorate + 2,4-diaminoanisole (DAAN), and (v) cells + iron treated PAX-21 wastewater. Each bottle was seeded with an initial cell concentration of 500 mg L−1. All experimental reactors were prepared in an anaerobic glove box filled with N2 gas (Bell-Art Products, Pequannock, NJ, USA). All bottles were placed on a rotary platform shaker (Lab-line, Melrose Park, IL, USA) in a horizontal position and continuously stirred at 150 rpm. At different elapsed times, about 2 mL of samples were taken from each bottle and passed through a 0.22-µm cellulose filter (Millipore) for chemical analysis.
Operation of integrated iron-bioreactor system An integrated iron-bioreactor system consisted of a glass column (1.5 cm ID × 20 cm L, Ace Glass, Vineland, NJ, USA) packed with Peerless iron granules (porosity = 0.65) and an anaerobic bioreactor system. An upflow anaerobic sludge bioreactor system for this study consisted of an acrylic column (3.81 cm ID × 80 cm long) and a 500mL Erlenmeyer flask. The acrylic column was filled with perchlorate-reducing cultures established from an activated sludge sample obtained from the Wilmington wastewater treatment plant (Wilmington, DE, USA). Glass beads were placed at the bottom of the acrylic column to distribute the flow evenly across the column cross-section. The nutrient solution was pumped from the 500-mL reservoir (300 mL liquid volume) using a Masterflex peristaltic pump (ColeParmer, Vernon Hills, IL, USA) to the bottom of the column bioreactor and the column effluent was returned to the reservoir from the top by gravity. A flow rate of 150 mL min−1 was determined as the optimum upflow rate for maintaining a constant sludge blanket height,[26] which allowed the recirculation of clear supernatant back to the reservoir. The nutrient reservoir also served as the mixing chamber to combine the iron-treated PAX-21 stream from the iron column with nutrient solution and recirculation stream. The constituents of nutrient solution were: 1.4 g L−1 Na2 HPO4 , 0. 9 g L−1 KH2 PO4 , 0.1 g L−1 (NH4 )2 SO4 , 0.2 g L−1 MgSO4 ·7H2 O, 1 mL trace mineral solution, and 1 mL Ca-Fe solution (adapted from Nerenberg et al. [24]). The Ca-Fe solution contained 1 g L−1 CaCl2 ·2H2 O, 1 g L−1 FeSO4 ·7H2 O. The trace mineral solution contained 100 mg L−1 ZnSO4 ·7H2 O, 30 mg L−1 MnCl2 ·4H2 O, 300 mg L−1 H3 BO3 , 200 mg/L CoCl2 ·6H2 O,
578 10 mg L−1 CuCl2 ·2H2 O, 10 mg L−1 NiCl2 ·6H2 O, 30 mg L−1 Na2 MoO4 ·2H2 O and 30 mg L−1 Na2 SeO3 . A second peristaltic pump (Cole-Palmer, Chicago, IL, USA) was used to supply the ZVI-treated PAX-21 wastewater to the nutrient reservoir at a rate of 0.1 L h−1. The treated effluent from the integrated system was withdrawn from the top of the bioreactor column at the same flow rate of 0.1 L h−1. Prior to a continuous feeding operation, the bioreactor system was operated in a batch-feeding mode for 4 weeks to establish acclimated perchlorate-reducing cultures. A control system receiving untreated PAX-21 wastewater was operated in parallel.
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Analytical methods Perchlorate was analyzed using a Dionex ICS-1000 ion chromatograph (Dionex, Sunnyvale, CA, USA) equipped with an IonPAC AS16 2-mm column, a AG16 2-mm guard column, and ASRS Ultra 2-mm suppressor. NaOH solution (65 mM) was used as the eluent and the injection volume was 25 µL. DNAN, 2-Methoxy-5-nitroanlinine, and 4Methoxy-3-nitroanlinine, RDX, DAAN and formaldehyde were analyzed using a Dionex HPLC (Dionex) equipped with a Supelguard LC-18 guard column (length, 20 mm; inner diameter, 4.0 mm; Supelco, Bellefonte, PA, USA) and a Supelcosil LC-18 column (length, 250 mm; inner diameter, 4.6 mm; film thickness, 5 mm). The mixture of methanol and deionized water (50/50, v/v) was used as an eluent at a flow rate of 1.0 mL/min for measuring DNAN, 2Methoxy-5-nitroanlinine, 4-Methoxy-3-nitroanlinine, and RDX. The wavelength for the UV detector was 275 nm for 2-Methoxy-5-nitroanlinine and 254 nm for DNAN, 4-Methoxy-3-nitroanlinine, and RDX. For DAAN, phosphate buffer (20mM, pH 7)-acetonitrile (70/30, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The wavelength for the UV detector was 275 nm. Formaldehyde concentrations were determined after derivatization with Nash’s reagent (0.02 M of 2,4-pentanedione and 2 M of ammonium acetate, pH 6).[27] One milliliter of aqueous formaldehyde sample was derivatized with 1 mL of Nash’s reagent for 1 h at 60◦ C in a water bath (Precision, Winchester, VA, USA). After the sample was cooled to room temperature (20 ± 2◦ C), concentrations of formaldehyde derivative were determined using the Dionex HPLC [wavelength for the UV detector 410 nm, eluent the mixture of acetonitrile and deionized water (70/30, v/v)]. The injection volumes for all samples were 250 µL. Ammonium ion was analyzed with the Hach spectrophotometer using the salicylate method (Hach, Loveland, CO, USA).
Results and discussion Iron reduction experiments PAX-21 wastewater obtained from Holston Army ammunition plant contained 49.5 mg L−1 of RDX, 123.6 mg L−1
Ahn et al. of DNAN, and 30.2 mg L−1 of perchlorate. Fig. 2 shows the concentrations of DNAN, reduction intermediates (2Methoxy-5-nitroanlinine and 4-Methoxy-3-nitroanlinine), the end product (DAAN), and total carbon during the reduction of PAX-21 wastewater with zero-valent iron (ZVI). DNAN was rapidly and completely removed by iron granules. Approximately 60% of the initial DNAN disappeared within 10 min and complete removal was achieved after 60 min. Less than 30% of reduced DNAN was recovered as intermediates in the first 30 min of reaction time. Among the reduction intermediates measured, 2Methoxy-5-nitroaniline was the dominant intermediate, indicating that reduction of the ortho nitro group was the primary reaction pathway for DNAN reduction by ZVI. Fig. 2 also shows that 100% carbon recovery was achieved in 2 h. The lower carbon recovery during the first 60 min may be due to adsorption of DNAN and reduction intermediates to iron surface, and accumulation of intermediates that were not measured in this study. Oh et al.[28] also reported that more than 70% of DNT removal in the first 10 min of reaction time was attributed to adsorption by iron granules. RDX in PAX-21 wastewater was also completely removed by ZVI granules in 2 h (data not shown). The batch removal rate of RDX in PAX-21 by ZVI was similar to RDX reduction rates reported by Oh et al.[28] The major end products of RDX reduction by zero-valent iron were formaldehyde (15.8 mg L−1) and NH4 + (12.5 mg L−1). The batch reduction experiments showed that DNAN and RDX in PAX-21 wastewater can be readily transformed to DAAN and formaldehyde by ZVI granules.
Batch biodegradation experiments with ZVI-treated PAX-21 wastewater To examine whether ZVI-treated energetic compounds in PAX-21 can serve as electron donor sources for perchloraterespiring bacteria, a series of batch biodegradation tests were conducted with iron-treated PAX-21 wastewater. PAX-21 wastewater was initially passed through an ironpacked glass column (2.5 cm ID × 30 cm L) at a flow rate corresponding to a 15-min contact time. ZVI column treatment resulted in complete removal of DNAN and RDX from PAX-21 wastewater and the column effluent contained 87.8 mg L−1 of DAAN, 20.5 mg L−1 of HCHO and 30 mg L−1 of perchlorate. Fig. 3 shows the disappearance of perchlorate in PAX-21 wastewater and ZVI-treated PAX-21 wastewater in batch bioreactors. Perchlorate in the iron-treated PAX-21 wastewater decreased to an undetectable level after 5 days of incubation in anaerobic bioreactors, yet negligible perchlorate was removed from reactors containing untreated PAX-21 wastewater. Without iron pre-treatment, the energetic compounds in PAX-21 wastewater was not able to support microbial perchlorate reduction; however, after ZVI treatment, complete removal of perchlorate was obtained without adding
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Experimental Days Fig. 6. Effluent perchlorate concentrations from ZVI-bioreactor system. Influent PAX-21 wastewater contained 30 mg L−1 perchlorate, 50 mg L−1 RDX, and 124 mg L−1 DNAN.
external sources of electron donors. This result clearly demonstrated that ZVI pretreatment not only removed energetic compounds, but also transformed energetic compounds to products that can serve as the source of electrons for perchlorate-respiring bacteria. To further validate that the microbial reduction of perchlorate can be coupled with ZVI-treated energetic compounds in PAX-21 wastewater, batch bioreactors containing perchlorate were spiked with iron-reduction products of RDX (formaldehyde) and DNAN (DAAN). Fig. 4 shows the disappearance of perchlorate in batch reactors containing 40 mg L−1 of formaldehyde. Aqueous perchlorate (30 mg L−1) began to decrease immediately and complete removal of perchlorate was achieved in 5 days of incubation. On the other hand, negligible amount of perchlorate was removed in 6 days of incubation in the control reactor containing no formaldehyde. This result suggested that formaldehyde can serve as the electron donor for the reduction of perchlorate by perchlorate respiration bacteria. Figure 5 shows the effect of DAAN addition (100 mg L−1) on microbial reduction of perchlorate by the same perchlorate-respiring bacteria. In contrast to formaldehyde, the addition of 100 mg/L DAAN did not support the reduction of perchlorate as the disappearance of aqueous perchlorate was similar to the removal rate observed in cell control. Because batch degradation experiments were carried out with non-acclimated activated sludge as seed cultures, the inability of DAAN to support microbial perchlorate reduction during this study may be due to the lack
of acclimated microorganisms. It has been reported that the microbial oxidation of aminoaromatic compounds proceeds only with acclimated cultures that typically require months for establishment.[29,30] Pesce and Wunderlin [30] reported activated sludge microorganisms required a 4-month acclimation period in batch reactors for the treatment of 2,4-DAT- and 2,6DAT-containing wastewaters. Since DAAN was not able to support microbial perchlorate reduction by the cultures used in this study, the removal of perchlorate (30.2 mg L−1) in the PAX-21 solution was most likely coupled with formaldehyde (20.5 mg L−1) from the RDX reduction reaction. Formaldehyde concentration in the iron-treated PAX-21 was greater than the stoichiometric amounts needed for the complete reduction of perchlorate in the solution. Operation of integrated ZVI-bioreactor system To demonstrate the application of ZVI technology for simultaneous removal of perchlorate and energetic compounds, PAX-21 wastewater was continuously treated with the integrated ZVI-bioreactor system. The integrated system was initially supplemented with the daily addition of acetate (150 mg L−1 NaCH3 COO) and perchlorate (30 mg L−1) for about 4 weeks to establish stable perchloratereducing populations in the upflow sludge blanket reactor. After 4 weeks of culture enrichment, daily addition of acetate and perchlorate was stopped and ZVI-treated
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PAX-21 wastewater containing about 30 mg L−1 perchlorate was continuously fed to the nutrient reservoir at 0.1 L h−1. Figure 6 compares the effluent perchlorate concentrations from the ZVI integrated system and control unit over the 14-day experimental period. Perchlorate concentrations in the effluent of the integrated system were consistently below the detection limit (0.02 mg L−1) (Fig. 6), while the perchlorate concentrations in the effluent of the control system without ZVI treatment were greater than 20 mg L−1. This study demonstrated that ZVI column transformed the energetic compounds in PAX-21 wastewater to products that supported the growth of an anaerobic culture, which was capable of completely reducing aqueous perchlorate.
Conclusions Results of batch experiments showed that DNAN was completely reduced to 2,4-diaminoanisole (DAAN) and RDX was completely reduced to formaldehyde in the presence of cast iron granules within 2 h. Perchlorate (30 mg/L) in ZVI-treated PAX-21 wastewater was decreased to an undetectable level after 5 days of incubation in a batch bioreactor containing seed microorganisms obtained from a wastewater treatment plant. Without ZVI pre-treatment, the energetic compounds in PAX-21 wastewater was not able to support microbial perchlorate reduction. Batch biodegradation experiments with ZVI-reduction products of energetic compounds showed that formaldehyde can serve as an electron donor for perchloraterespiring bacteria. On the other hand, DNAN reduction product, DAAN, was not able to support the perchloratereducing activity by PRB. Anaerobic batch experiments demonstrated that complete reduction of perchlorate in iron-treated PAX-21 wastewater can be achieved without adding an exogenous electron donor. The integrated ZVIanaerobic bioreactor system was effective in removing perchlorate from PAX-21 wastewater without the addition of external electron donors. This study demonstrated that ZVI pretreatment not only removed energetic compounds, but also transformed the energetic compounds to products that can serve as the source of electrons for perchlorate-respiring bacteria. Use of zero-valent iron may eliminate the need to continually supply electron donors such as organic substrates to remove perchlorate.
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