Remediation of Polycyclic Aromatic Hydrocarbons

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Introduction. Creosote oil was used decades ago to extend the life of railroad timber as well as electric power poles (Bolin and Smith 2013). A previous study reported that creosote oil contains over 75–80% .... Therefore, the presence of hemoglobin did not affect the ..... Rentz, J. A., Alvarez, P. J. J., and Schnoor, J. L. (2008).

Remediation of Polycyclic Aromatic Hydrocarbons in Soil Using Hemoglobin-Catalytic Mechanism

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Guyoung Kang 1; Kapsung Park 2; Jaechang Cho 3; David K. Stevens, F.ASCE 4; and Namhyun Chung 5

Abstract: It is suggested in this paper that recalcitrant organopollutants can be degraded efficiently by a hemoglobin-catalytic reaction in the presence of hydrogen peroxide (i.e., H2 O2 ). The catalytic mechanism was studied with 5-aminosalicylic acid (5-ASA) as a compound for oxidation. Various evidence suggests that the catalytic mechanism is very similar to those of horseradish peroxidase and lignin peroxidase. The catalytic intermediates are known to oxidize various chemicals, indicating that the intermediates of hemoglobin can nonspecifically degrade many different types of organopollutants. To prove the hypothesis, an attempt was made to remediate polycyclic aromatic hydrocarbon (PAH)-contaminated field soil. The results showed that 98.5% of the PAHs compounds were removed by Day 42 and that seven of the 16 PAHs compounds analyzed were not detectable by the end of the research reported in this paper. Therefore, hemoglobin-catalyzed technology can be considered as a novel technology for remediation of soil contaminated with hazardous organopollutants. DOI: 10.1061/ (ASCE)EE.1943-7870.0000955. © 2015 American Society of Civil Engineers. Author keywords: Hemoglobin; Catalytic mechanism; Remediation; Hydrogen peroxide; Polycyclic aromatic hydrocarbons.

Introduction Creosote oil was used decades ago to extend the life of railroad timber as well as electric power poles (Bolin and Smith 2013). A previous study reported that creosote oil contains over 75–80% polycyclic aromatic hydrocarbon (PAH) compounds and that approximately 2.74 Gkg of railroad timber was used for over 130 years in North America. Pentachlorophenol (PCP) and creosote oil was widely used for bomb wood boxes as well as railroad timber and electric power poles before Year 1900 (Crosby 1981; Heikkila et al. 1987). Therefore, the soil around the timber, poles, and boxes can be expected to be contaminated with PAHs and PCP (Bolin and Smith 2013; Chen et al. 1999; Kang et al. 1995). There has been increasing interest in abiotic and biotic processes for the destruction of hazardous PAH compounds in contaminated soil. Most PAHs compounds are toxic to humans and animals [Agency for Toxic Substances and Disease Registry (ATSDR) 1995; Menzie et al. 1992; Wood et al. 1976]. For example, benzo½apyrene is considered carcinogenic and was included in the hazardous materials list according to Occupational Safety and Health Administration (OSHA) guideline 29 CFR 1910.1200. In an effort to degrade PAHs compounds in soil, many biotic processes were developed. However, bacteria or bacterial consortia 1

Professor, Dept. of Environmental Science, Hankuk Univ. of Foreign Studies, Yongin 449-791, Korea. E-mail: [email protected] 2 Professor, Dept. of Environmental Science, Hankuk Univ. of Foreign Studies, Yongin 449-791, Korea. E-mail: [email protected] 3 Professor, Dept. of Environmental Science, Hankuk Univ. of Foreign Studies, Yongin 449-791, Korea. E-mail: [email protected] 4 Professor, Dept. of Civil and Environmental Engineering, Utah State Univ., Logan, UT 84321. E-mail: [email protected] 5 Professor, College of Life Sciences and Biotechnology, Korea Univ., Seoul 136-713, Korea (corresponding author). E-mail: [email protected] .kr; [email protected] Note. This manuscript was submitted on September 19, 2014; approved on January 23, 2015; published online on March 23, 2015. Discussion period open until August 23, 2015; separate discussions must be submitted for individual papers. This paper is part of the Journal of Environmental Engineering, © ASCE, ISSN 0733-9372/04015025(5)/$25.00. © ASCE

have a limiting degree of degradation in a highly contaminated soil. Additionally, they are required to be adapted to the specific chemicals of interest and to have supplementary nutrients (Bengtsson and Zerhouni 2003; Fernández-Luque˜no et al. 2011; Rentz et al. 2008). Barr and Aust (1994) reported that lignin-degrading white fungi are able to degrade lignin and many hazardous organic pollutants simultaneously through the peroxidase catalytic cycle. Regardless of many strong points, the ligninolytic fungi should be under a severe competition with common soil microbial communities, making the white-rot fungi unsuitable for a bioremediation in situ (Hadibarata 2009; Lamar and Evans 1993). In another effort to remediate PAHs, many abiotic processes including Fenton reaction have been suggested as a solution. However, Fenton reaction requires both a slurry reactor and a low pH environment, making it uneconomical for in situ application (Jonsson et al. 2007; Nadarajah et al. 2002; Nam et al. 2001). Numerous scientists have shown that organopollutants including PAHs, PCP, crystal violet, and dibenzothiophen can be degraded by heme and hemoglobin in the presence of hydrogen peroxide (Chen et al. 1999, 2006; Kang et al. 1995; Ortiz-Leon et al. 1995; Stachyra et al. 1996). For example, Ortiz-Leon et al. (1995) reported that hemoglobin can oxidize a limited number of PAHs in the presence of hydrogen peroxide under solution containing solvent. In this paper the development of soil remediation technology is reported, employing dry hemoglobin to degrade PAH compounds in soil. Objectives of the research reported in this paper are to prove the hemoglobin catalytic mechanism using 5-aminosalicylic acid (5-ASA) as a model organic chemical, and to prove the degradation of PAHs in soil in the presence of hemoglobin and hydrogen peroxide. Thus, the catalytic mechanism and the abiotic degradation of PAHs in contaminated soil were assessed.

Experimental Section Chemicals and Materials Powder hemoglobin from bovine blood, 5-aminosalicylic acid, 30% hydrogen peroxide, and trichloroacetic acid (TCA) were

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purchased from Sigma–Aldrich (St. Louis, Missouri). Powder hemoglobin was donated by American Laboratories (Omaha, Nebraska) for use in the pan study for remediation of PAHcontaminated soil. Other reagent grade chemicals were purchased from Duksan Pharmaceutical (Ansan, Gyeonggi-do, Korea). The hemoglobin stock solution (38 μM) was prepared before each experiment from powder hemoglobin by dissolving 24.2 mg in 10 mL 50 mM phosphate buffer (pH 7.0). An aliquot (25 μL) of hydrogen peroxide (0.015 μM) was injected into 3 mL of the reaction mixture with a calibrated disposable micropipette (VWR, Radnor, Pennsylvania) and 5% trichloroacetic acid was dissolved in 50 mM phosphate buffer. Spectral Characterization of Hemoglobin-Catalytic Reaction

Table 1. Composition of Catalytic Reaction Mixture Three-mL total volume in quartz cuvette Task number 1 2 3 4

a

Spectrum, photometric, and kinetic measurements for the hemoglobin-catalytic reaction were obtained using a UV-visible spectrophotometer (UV-1650PC, Shimadzu, Tokyo, Japan). All catalytic reaction mixtures contained 12.5 μM hemoglobin, 50 mM phosphate buffer (pH 7.0), 100 μM 5-ASA, and 125 μM H2 O2 (3 mL total volume) in a quartz cuvette (Starna Scientific, Essex, U.K.) at 24  2°C. Catalytic reactions were initiated by addition of 25 μL hydrogen peroxide (for a final concentration of 125 μM). The oxidation and reduction states of hemoglobin were observed by scanning at wavelengths between 250 and 700 nm. For the hemoglobin-catalytic mechanism, the ability of hemoglobin to catalyze the H2 O2 -dependent oxidation of 5-ASA (100 μM) was evaluated. The catalytic reactions were terminated by adding 1 mL solution containing 5% (weight by volume) TCA and vortexing for 10 s. Coagulated hemoglobin was removed from the reaction mixture by filtration using 0.2-μm filters (Acrodose LC 13 polyvinylidene difluoride, Gelman Sciences, Ann Arbor, Michigan), and the filtered mixture was scanned in a 1-mL QS quartz cuvette (Hellma Analytics, Müllheim, Germany). Degradation of PAH-Contaminated Soil by Hemoglobin and Hydrogen Peroxide The pan study was performed with pole yard soil obtained from Washington State. The PAH-contaminated soil contained 464 mg of total PAHs=kg soil. Five hundred grams of pole yard soil (a silt loam soil with a ratio of sand:silt:clay = 23:60:17%; organic carbon, 2.85%; pH 5.5; field capacity, 33%) was placed in a stainless steel pan, and 8.8 g dry hemoglobin was added and mixed. Next, 81 g H2 O2 in 50 mM phosphate buffer (pH 7.0) was diluted with distilled water to a final volume of 250 mL and mixed well by spraying. The 50% moisture was maintained by adding 50 mM phosphate buffer (pH 7.0) during the treatment. PAHs samples were extracted using a Soxhlet device [U.S. EPA Method 3540C (U.S. EPA 1996)] and subjected to an acid/base cleanup according to U.S. EPA Method 3650A (U.S. EPA 1992). The PAH samples were analyzed using HPLC (SIL-6B, Shimadzu) with a UV detector (LC 90UV Spectrophotometric, Perkin–Elmer, Waltham, Massachusetts).

Componentsa

Stock solution (μM)

Injection volume (μL)

Final concentration (μM)

5-ASA Hemoglobin H2 O2 Hemoglobin 5-ASA Hemoglobin 5-ASA H2 O2

300 38 15,000 38 300 38 300 15,000

1,000 1,000 25 1,000 1,000 1,000 1,000 25

100 12.5 125 12.5 100 12.5 100 125

5-ASA = 5-aminosalicylic acid.

phosphate buffer, 100 μM 5-ASA, and/or 125 μM H2 O2 in a total volume of 3 mL. Spectral characteristics were determined by scanning wavelengths between 250 and 700 nm after mixing in the cuvette for 1 min. However, the maximum absorbance of hemoglobin at 400 nm was large enough to interfere with the observed change in the concentration of oxidized 5-ASA. As a result, we were unable to detect the oxidized form of 5-ASA, which was used as a model organic compound in the research reported in this paper. The previously noted experiment was therefore retried after hemoglobin was removed by filtration after the addition of 1 mL 5% TCA. Fig. 1 illustrates the UV/visible absorption spectrum of filtrates obtained after the reaction of 5-ASA and/or hemoglobin in the presence or absence of H2 O2 at pH 7.0. During this entire reaction, hemoglobin had been completely filtered out after adding 5% TCA. Therefore, the presence of hemoglobin did not affect the absorbance spectrum of 5-ASA without H2 O2 (Fig. 1, Curves A and C). When hemoglobin and H2 O2 were both present, the spectrum changed into Curve B (Fig. 1). When hemoglobin and 5-ASA were both initially present, with H2 O2 added later, the spectrum changed and the absorbance at 400 nm increased significantly. The peak at 400 nm is specific for the oxidized 5-ASA (5-ASAox ) as has been reported by Yamada et al. (1991). The phenomenon

Results and Discussion Oxidation of 5-ASA by Hemoglobin-Catalytic Reaction Components of the mixed solution to study catalytic reactions are presented in Table 1. UV-visible spectra analyses were obtained to assay hemoglobin interaction with H2 O2 and 5-ASA. All catalytic reaction mixtures contained 12.5 μM hemoglobin in 50 mM © ASCE

Fig. 1. Absorption spectrum of oxidized 5-ASA after removal of hemoglobin; Curve A represents the absorption spectrum of 5-ASA; Curve B represents the spectrum obtained with hemoglobin and H2 O2 ; Curve C represents the spectrum obtained hemoglobin and 5-ASA (no added H2 O2 ); Curve D represents the spectrum obtained after addition of H2 O2 to hemoglobin and 5-ASA

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Fig. 2. Time-dependent oxidation of 5-ASA; changes in absorbance (400 nm) for 5-ASAox were observed after removal of hemoglobin; initial oxidized 5-ASA was measured without H2 O2

may be due to the catalytic mechanism of hemoglobin as discussed in a subsequent paragraph. Compound 5-ASA was not oxidized without either hemoglobin (data not shown) or H2 O2 (Fig. 1; Curve C). Hemoglobin-Catalytic Mechanism Based on the previous result, we decided to determine the level of the formation of oxidized 5-ASA by measuring the absorbance at 400 nm for 4 min. The oxidation of 5-ASA was measured after removal of hemoglobin at the end of the reaction as described previously in this paper (Fig. 2). The result shows that 5-ASA was oxidized by hemoglobin-catalytic reactions for 4 min. In the hemoglobin-catalytic reactions, hemoglobin (Hb-Feþ3 ) may be rapidly converted to the ferryl-state hemoglobin radical (Hb-Feþ4• ) in the presence of H2 O2 . The ferryl-state hemoglobin is then reduced by 5-ASA while 5-ASA becomes oxidized. To prove this hypothesis, the formation and disappearance of ferrylstate hemoglobin (Hb-Feþ4 ) were observed in reaction mixtures containing 5 μM hemoglobin only, 5 μM hemoglobin plus 50 μM H2 O2 , or 5 μM hemoglobin plus 50 μM H2 O2 with the addition of 400 μM 5-ASA (Fig. 3). Addition of H2 O2 to hemoglobin resulted in the formation of a ferryl-state hemoglobin (Hb-Feþ4 ; Fig. 3; Curve B), which has visible peaks at both 545 and 576 nm as shown by Yamada et al. (1991). When 5-ASA was added to the preformed ferryl-state hemoglobin (Hb-Feþ4 ), the absorbance at the two specified peaks decreased significantly (Fig. 3; Curve C), suggesting the reduction of the ferryl-state hemoglobin (Hb-Feþ4 ) to the original ferric-state hemoglobin (Hb-Feþ3 ). The time-dependent formation of ferryl-state hemoglobin was observed with a visible peak at 545 nm from ferric-state hemoglobin (Hb-Feþ3 ) in the presence of H2 O2 (Fig. 4). When 5-ASA was added to the preformed ferryl-state hemoglobin (Hb-Feþ4 ) at 120 s, a gradual reduction in absorbance was observed at 545 nm, suggesting that 5-ASA was being oxidized by ferryl-state hemoglobin (Hb-Feþ4 ), which returned to ferric-state hemoglobin (Hb-Feþ3 ). Hemoglobin has a strong absorbance at 405 nm (i.e., Soret band or peak) but the absorbance decreases significantly with formation of ferryl-state hemoglobin (Hb-Feþ4 ) in the presence of H2 O2 (Yamada et al. 1991). Using this phenomenon, the conversion of ferryl-state hemoglobin (Hb-Feþ4 ) to ferric-state hemoglobin (Hb-Feþ3 ) in the presence of 5-ASA was observed (Fig. 5). In the absence of H2 O2 , the absorbance did not change (Fig. 5; Curve A). © ASCE

Fig. 3. Reduction of ferryl-state hemoglobin (Hb-Feþ4 ) by 5-ASA as observed with change in the visible band of hemoglobin; Curve A represents the absorption spectrum obtained with 5 μM hemoglobin (Hb-Feþ3 ) in 50 mM phosphate buffer (pH 7.0); Curve B represents the spectrum obtained with hemoglobin (Hb-Feþ3 ) and 50 μM H2 O2 ; Curve C represents the spectrum obtained after addition of 400 μM 5-ASA to the solution containing hemoglobin (Hb-Feþ3 ) and 50 μM H2 O2

In the presence of H2 O2 , the absorbance decreased rapidly with time. However, the decrease in absorbance slowed with addition of 5-ASA. At the end of the 120-s observation period, the absorbance at 405 nm with 5-ASA (Fig. 5; Curve C) was much higher than without 5-ASA (Fig. 5; Curve B). This suggests that most, but not all, of the ferryl-state hemoglobin (Hb-Feþ4 ) is reduced back to ferric-state hemoglobin (Hb-Feþ3 ), completing the hemoglobin catalytic cycle. The 5-ASA in this reaction might behave as a reactant and is oxidized to the 5-ASA radical, which can be further oxidized to 5-ASAox . That is, hemoglobin uses H2 O2 to promote the two-electron oxidation of ferric-state hemoglobin to ferryl-state hemoglobin radicals (Hb-Feþ4• ), which is rapidly reduced to ferryl-state hemoglobin (Hb-Feþ4 ) with one-electron oxidation of 5-ASA. The ferrylstate hemoglobin (Hb-Feþ4 ) returns to ferric-state hemoglobin

Fig. 4. Time-dependent change in absorbance at the visible band (545 nm); ferryl-state hemoglobin (Hb-Feþ4 ) was formed by adding H2 O2 to ferric-state hemoglobin (Hb-Feþ4 ); after 120 s, 5-ASA was added to the reaction mixture and the change in absorbance was monitored; reaction mixtures were hemoglobin, H2 O2 , or 5-ASA

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Fig. 5. Time-dependent change in absorbance at Soret peak (405 nm); reaction mixtures were 5 μM hemoglobin, 50 μM H2 O2 , or 400 μM 5-ASA in 50 mM phosphate buffer (pH 7.0); Curve A represents the absorption spectrum of hemoglobin (Hb-Feþ3 ); Curve B represents the spectrum obtained with hemoglobin (Hb-Feþ3 ) and H2 O2 ; Curve C represents the spectrum obtained with 5-ASA, hemoglobin (Hb-Feþ3 ), and H2 O2

(Hb-Feþ3 ) by one-electron oxidation of 5-ASA or 5-ASA radical, which is converted to 5-ASAox eventually. This kind of catalytic cycle has also been reported with lignin peroxidase in white-rot fungus (Barr and Aust 1994). Based on these results, the hemoglobin catalytic reaction mechanism is summarized as shown in Fig. 6. The mechanism is similar to the previously proposed mechanism with regards to the remediation of pentachlorophenol-contaminated soil (Chen et al. 1999, 2006; Kang et al. 1995). Degradation of PAH in Soil Based on the catalytic mechanism, the remediation of PAHcontaminated soil was tested. Polycyclic aromatic hydrocarbon

Fig. 7. Time course for degradation of PAHs in pole yard soil treated with hemoglobin and hydrogen peroxide; controls were treated with buffer or H2 O2

compounds are as a component of the creosote oil once used for wood preservation. The 2.6 and 12.9% of total PAH compounds were degraded for controls with buffer and H2 O2 , respectively (Fig. 7). Total PAH compounds of 2.7% were degraded as a control with hemoglobin (data not shown). However, in soil treated with hemoglobin and H2 O2 together, PAHs concentration decreased from 464 to 194 mg=kg soil at Day 14 and further decreased to 7.2 mg=kg soil at Day 42 (Table 2; Fig. 7). Thus, about 98.5% of PAHs compounds were degraded in 42 days. This result indicated that the cotreatment of dry hemoglobin and hydrogen peroxide can be used for remediation of PAH-contaminated soil. In the research reported in this paper, the catalytic activity is believed to be performed with heme group upon reacting with H2 O2 as it has been suggested in previous studies with heme (Chen et al. 1999, 2006). Then, the role of protein portion of hemoglobin except heme group is questioned. It is suggested in this paper that, after initial PAH degradation activity is performed in the presence

Table 2. Degradation of PAHs in Pole Yard Soil by Hemoglobin and Hydrogen Peroxide in the Pan Study Polycyclic aromatic hydrocarbon compounds

Fig. 6. Hemoglobin catalytic cycling in the presence of hydrogen peroxide and reactant (RH); Hb-Feþ4• indicared ferryl-state hemoglobin radical, Hb-Feþ4 indicates ferryl-state hemoglobin, Hb-Feþ3 indicates ferric-state hemoglobin, R• indicates reactant radical, and Rox indicates oxidized reactant; Rox could be oxidized further to be mineralized into CO2 © ASCE

Naphthalene Acenaphthalene Acenaphthene Fluorene Pheneanthrene Anthracene Fluranthene Pyrene Benzo½aanthracene Chrysene Benzo½bfluoranthene Benzo½kfluoranthene Benzo½apyrene Dibenzo½a; hanthracene Benzo½g; h; iperylene Ideno½1; 2; 3cdpyrene Total PAHs a

Operation time in the pan study (mg=kg soil)a Day 0

Day 14

Day 42

49.32  4.38 16.66  5.14 16.64  5.74 2.94  0.25 102.78  17.50 7.26  1.27 3.08  2.02 4.04  1.64 11.30  3.02 4.20  1.98 5.24  1.54 ND 111.84  3.81 60.40  5.05 73.40  9.00 37.54  3.54 24.62  5.37 37.40  7.42 28.28  3.04 9.82  2.33 12.28  0.98 3.68  1.11 5.54  1.89 4.66  0.82 5.38  2.12 0.70  0.12 3.00  1.40 0.92  0.12 8.70  1.78 2.08  0.21 0.32  0.08 3.26  0.54 463.78  63.06 193.50  32.15

ND ND ND ND ND ND 0.40  0.10 3.10  1.12 0.46  0.02 0.06  0.01 0.34  0.09 2.38  0.54 0.12  0.03 0.18  0.03 ND 0.16  0.04 7.20  1.98

Values are presented as mean  SD. ND = not detectable.

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of H2 O2 , the remaining protein portion is subject to microbial degradation, helping continued degradation of PAH compounds. Because the results of this paper suggest that the hemoglobin-catalytic reaction is similar to that of lignin peroxidase (Barr and Aust 1994), reaction in the presence of H2 O2 may continue as long as H2 O2 is present in soil. A while after the added amount of H2 O2 is consumed, endogenous soil microbes may continue to grow after adaptation to the treated environment. It has been estimated for this paper that it takes about 1 or 2 weeks for soil microbial communities to be adapted to the treated soil environment. Considering all these facts, the first PAH degradation was measured in 14 days as shown in Fig. 7. It may need further efforts to decide how long the catalytic reaction continues in soil and how long it takes for soil microbes to be adapted to the treated soil environment. The results of previous works have demonstrated that treatment of soil containing the wood preservative PCP with heme and hydrogen peroxide induced a significant decrease in the PCP concentration under both laboratory and field conditions using pole yard soil obtained from Vancouver, Washington (Chen et al. 2006). Removal of up to 80% of PCP was observed within 4 h after treatment with 8.5 g heme and 47.5 g hydrogen peroxide per kilogram soil. Together with previous results with PCP, the present results indicate that hemoglobin-catalytic mechanism is able to degrade a variety of hydrophobic organic contaminants in soil. This abiotic remediation technology has many advantages. Unlike other biotic processes, the technology has no need of adaptation to the environment (versus bacterial degradation technology) or competition with soil microbial communities (versus white-rot fungi technology). When compared with Fenton reaction, the technology could be employed in situ since it does not need a slurry condition or a low pH environment. Additionally, the price of hemoglobin is reasonably cheap (www.sztaier .com, Shenzhen Taier Biotechnology, Shenzhen, Guangdong 518052, China). The writers believe that this new remediation technology can be one of the most dependable technologies for the remediation of hazardous organopollutants in soil.

Conclusions The research reported in this paper demonstrated the hemoglobincatalytic reaction mechanism using 5-ASA as a compound for oxidation in the presence of hydrogen peroxide. On the whole, it is believed that the hemoglobin-catalytic reaction resembles that of horse radish peroxidase and lignin peroxidase. In addition, PAHscontaminated pole yard soil was remediated up to 98.5% by the hemoglobin catalytic reaction. In particular, benzo½apyrene, a carcinogenic component of PAHs, was not detected by Day 42, suggesting that the hemoglobin-catalytic reaction is very efficient. It is suspected that catalytic reactions involving hemoglobin and H2 O2 may also be useful for the remediation of many other organopollutants in soil.

Acknowledgments The research reported in this paper was supported by the Korea Ministry of Environment (MOE) as Geo- Advanced Innovative Action Project Program.

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