Science of the Total Environment 435–436 (2012) 563–566

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Science of the Total Environment

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Short Communication

Integrated hybrid treatment for the remediation of2,3,7,8-tetrachlorodibenzo-p-dioxin

Varima Bokare, Kumarasamy Murugesan, Jae-Hwan Kim, Eun-Ju Kim, Yoon-Seok Chang ⁎School of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, 790‐784, South Korea

H I G H L I G H T S

► Facile synthesis of bimetallic Pd/nFe nanoparticles was carried out in the laboratory.► 2,3,7,8-TeCDD was completely degraded at ambient conditions by Pd/nFe nanoparticles.► DD was the sole end product obtained after reduction of 2,3,7,8-TeCDD by Pd/nFe.► DD oxidation was achieved by sequential biodegradation using Sphingomonas wittichii.

⁎ Corresponding author. Tel.: +82 54 279 2281; fax:E-mail address: yschang@postech.ac.kr (Y.-S. Chang

0048-9697/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.scitotenv.2012.07.079

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 April 2012Received in revised form 15 July 2012Accepted 22 July 2012Available online 19 August 2012

Keywords:2,3,7,8-TeCDDBimetallic nanoparticlesReductionBiomineralizationNano-bio redox processHybrid treatment

The dioxin isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) has been reported as the deadliest com-pound known to science. Due to its highly recalcitrant nature and low bioavailability, it is stubborn toward biore-mediation and chemical treatment. Efforts to degrade it using one single technique have not accomplished thedesired results. In this study, we have tried to develop an integrated 2,3,7,8-TeCDD removal process usingpalladized iron nanoparticles (Pd/nFe) for initial reductive dechlorination under anoxic conditions and subse-quent oxidative biomineralization. Using laboratory synthesized Pd/nFe, 2,3,7,8-TeCDD was completelydechlorinated to form the end product dibenzo-p-dioxin (DD). Oxidative degradation of DD was successfullyachieved by growing active cells of a dioxin-degrading microorganism Sphingomonas wittichii RW1 (DSM6014) under aerobic culture conditions. Metabolite identification was done by high performance liquid chroma-tography (HPLC) and whole cell protein was measured as the indicator for cell growth. To the best of our knowl-edge, this is the first report on integrated hybrid degradation method for 2,3,7,8-TeCDD.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Polychlorinated dibenzo-p-dioxins (PCDDs) are highly toxic, sta-ble, persistent and chemically inert compounds (Park et al., 2011)that have attracted widespread public awareness due to their severeeffects on human health. Among the 75 PCDD congeners, only 7have substituted chlorines at the 2nd, 3rd, 7th and 8th position,which confers them with the highest toxicity (Kulkarni et al., 2008).They are commonly referred to as dioxins and came into the publiceye for the first time in 1982 due to the explosion at the ICMESA fac-tory in Seveso, Italy. These compounds are also produced naturallyduring volcanic eruptions, as by-products of combustion and in forestfires (Chang, 2008). They can also be formed during the synthesis ofchlorinated herbicides and pesticides; and also in the pulp andpaper manufacturing industry (Chang, 2008). Release of dioxins intothe atmosphere due to various chemical, industrial and thermal pro-cesses poses great threat to flora and fauna. The 2,3,7,8-(laterally)substituted congener (TeCDD) is extremely resistant to degradation.

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Recently, this compound garnered great public attention, after itsalleged role in the poisoning of Viktor Andriyovych Yushchenko,the former President of Ukraine (Sorg et al., 2009). This compoundis also a contaminant in Agent Orange which was used by the U.S.army during the VietnamWar. Due to its immunotoxic, neurotoxic,carcinogenic nature and planar structure, it is the deadliest com-pound known to science with a TEF (toxicity equivalence factor)of 1.

1.1. Limitations of the existing remediation technologies

TeCDD is highly persistent and resistant to degradation, makingeradication very challenging. No single technology can fully destroythis hazardous compound under ambient conditions. Incineration, bio-remediation and chemical treatment have been ineffective in removingTeCDD from environmental matrices. Due to the poor bioavailability ofthis compound, biodegradation is also slow and virtually ineffective.Oxidative removal by the Fenton process (Fe2++H2O2) has shown suc-cess, but addition of high amounts of hydrogen peroxide is not cost ef-fective for actual field remediation (Kao and Wu, 2000). Moreover,H2O2 is toxic to microorganisms, and the use of this oxidantmay inhibit

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natural bioremediation. Also, the most significant bottleneck of theFenton process is the requirement of strict acidic conditions whichare not always found in groundwater and aquifers. The sub-criticalwater treatment process is effective but requires high temperatures(>350 °C) and is therefore not practically viable. Although thephotodegradation of low concentrations of TeCDD present in herbi-cide formulations was very successful under sunlight (Crosby andWong, 1977), the degradation of pure crystalline TeCDD was verydifficult. The use of UV light in conjunction with an electron donormakes this process uneconomical under field conditions. Radiolyticdegradation and dechlorination using alkaline polyethylene glyconates(APEG-PLUS) have been reported (Hllarides and Gray, 1994), but thestrict reaction conditions necessary and high initial energy inputs ren-der these methods unfit for actual onsite soil/water treatment. Overall,TeCDD degradation methods that are cost-effective and do not requirestrict reaction conditions or externally added supplements are currentlynot available. Thus, our group has focused on developing a treatmentprocess for the rapid and effective degradation of TeCDD under mildconditions.

1.2. Nano-metal catalyst and dioxin degrading microorganisms

For decades, scientists have been trying to develop the “ultimate”catalyst that functions as a “One-Solution-to-All-Pollution”. Becausesuch efforts require benign, effective and durable decontaminants,zero-valent metals are potential candidates to fulfill this role. Zero-valent iron nanoparticles (nFe) have shown immense potential forthe destruction of numerous toxic compounds. By using a smallamount (b0.5 wt.%) of a noble metal such as platinum or palladium,potent bimetallic nanocatalysts with a core-shell formation (ironcore with Pd or Pt shell) have emerged as effective redox catalyststo degrade recalcitrant contaminants. Over the past 5 years, our re-search group has developed an expertise on the synthesis of nFeand Pd/nFe nanoparticles. Using iron based nanomaterials, we havedeveloped a process for the complete destruction of dioxins (Kim etal., 2008). However, not all hydrophobic compounds, such as dioxins,can be destroyed fully by nanoparticles alone. In the past decade, wealso have performed biodegradation studies on dioxins such as2,7-dichloro-p-dioxin, 1,2,3,4-tetrachlorodibenzo-p-dioxin, 1,2,3-tri-chlorodibenzo-p-dioxin and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin tounderstand the biotransformation kinetics and metabolite profileusing Sphingomonas wittichii RW1 (Hong et al., 2002; Nam et al.,2006). However, degradation rates were extremely slow, and completetransformation into lower congeners was not achieved even after longincubation times under optimum conditions.

Hybrid technologies have gained great importance in recent years.A nano-bio redox system involving anaerobic dechlorination usingnanoparticles in easily biodegradable congeners and subsequent min-eralization by bacteria or fungal enzymes under aerobic conditionshas been developed (Bokare et al., 2010). The combination of twohighly promising remediation technologies (nanotechnology and bio-technology) is an efficient method used to remove noxious sub-stances that are highly stubborn to degradation. Using this coreprinciple, our group has attempted to design a hybrid redox processthat incorporates reductive TeCDD dechlorination by Pd/nFe underanaerobic conditions and subsequent oxidative degradation of thenon-chlorinated product (DD) using the dioxin-degrading microor-ganism Sphingomonas wittichii RW1 (DSM 6014).

2. Materials and method

2.1. Chemicals

The following compounds were used as received: MCDDand 2,3,7,8-TeCDD (>99.9%, Accustandard), dibenzofuran (DF,Sigma-Aldrich), toluene (Merck), tetrahydrofuran (Merck),

iron(III) chloride (FeCl3.6H2O, Merck, UK), sodium borohydride(NaBH4, Merck) and palladium acetate (Pd(COOCH3)2, Aldrich).The synthesis and characterization of the Pd/nFe nanoparticles havebeen described by Kim et al. (2008). All reagents and solutionswere prepared in ultra-pure water (18 MΩ/cm). Sphingomonaswittichii RW1 (DSM 6014) was obtained from DSMZ, Germany andwas routinely maintained using DF as the sole carbon source.

2.2. Batch experiments

Since 2,3,7,8-TeCDD is a highly toxic compound, all experimentswere conducted in a special facility equipped with safety gears. Ex-periments were performed in 50 mL glass reactors with 30 mL ofliquid volume. In each batch bottle, 0.5 g of nanoparticles wasadded. 29 nmol of 2,3,7,8-TeCDD was spiked from the THF stock so-lution. THF was evaporated under a stream of nitrogen and 20 mLof degassed deionized water (pH adjusted to 2.5 by 1 N perchloricacid) was added in each reactor. The reactors were purged for5 min and sealed immediately and placed in a rolling mixer(40 r.p.m.) at an ambient temperature. Appropriate controls werekept and the experimentwas conducted in duplicates. After the desiredreaction time, samples were extracted and analyzed for 2,3,7,8-TeCDDand its dechlorinated products.

2.3. Extraction and analysis

A modified liquid–liquid extraction procedure was used in thisstudy. At each sampling time, duplicate reactors were taken and con-centrated HCl was added to dissolve all the nanoparticles, which en-abled adsorbed 2,3,7,8-TeCDD and dechlorinated products to bereleased from the metal surface (Kim and Carraway, 2003). The mix-tures were extracted twice with toluene by vigorous hand‐shakingfor 2 min and followed by sonication for 15 min (Hadnagy et al.,2007). The extracted layers were pooled in glass vials. Traces ofmoisture in the extracts were removed by adding sodium sulfate. Aportion of the extract was transferred to GC vials and analyzed. Theidentification of chlorinated end products of TeCDD degradationwith Pd/nFe was performed by gas chromatograph/mass spectrome-try (GC-MSD, Agilent 7890A/MSD Agilent Technologies 5475 C witha triple axis detector and an inert XLEI/CI mode) with a DB-5MS col-umn (30 m, 0.25 mm i.d., 0.25 μm film thickness). 1 μL of the samplewas injected in split-less mode with the inlet maintained at 280 °C.The oven temperature of GC was as follows; (Bokare et al., 2010)140 °C for 4 min; (Chang, 2008) increased at 15 °C/min to 220 °C;(Crosby and Wong, 1977) increased at 1.5 °C/min to 240 °C for2 min; (Hadnagy et al., 2007) increased at 4 °C/min to 310 °C for6 min. Helium was used as the carrier gas.

2.4. Biodegradation of DD

The aqueous solution containing DD as the end-product of TeCDDdechlorination was inoculated with active cells of strain RW1 (grownwith DF as the substrate) at cell concentrations of 0.01–0.02 OD (Abs600 nm) and then incubated at 30 °C at 160 r.p.m. At different timeintervals, samples were collected in duplicates and analyzed for DDdegradation, metabolite identification and bacterial growth. To con-firm the degradation process, a comparative experiment was alsoperformed with an equal amount of DF and monochloro‐dibenzo-p-dioxin (MCDD). Analysis of the residual substrate and metaboliteidentification were done in a HPLC system (Agilent 1100) fittedwith Zorbox C18 column using acetonitrile and 0.1% phosphoric acid(60:40 v/v) as the eluents. The whole cell protein content was mea-sured for bacterial growth.

Fig. 2. Biomineralization of DD by bacteria ([RW1 at 0.01600 nm, 30 °C], [DD]0=27 nmoland pHi=7).

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3. Result and discussion

3.1. Dechlorination of 2,3,7,8-TeCDD

Under anaerobic conditions and at acidic pH values, complete de-chlorination of 2,3,7,8-TeCDD was observed within 10 h by Pd/nFe(0.5 g). The time profile of TeCDD degradation is shown in Fig. 1. Assoon as the nanoparticles come in contact with TeCDD, the dechlori-nation reaction is initiated, with a 30–40% decrease in TeCDD concen-tration initiated within 1–2 h of the reaction (Fig. 1). GC-MS studiesshowed the generation of mono-CDDs to tri-CDDs within 2–6 h,which were also eventually degraded to form the major end productDD. The dechlorination process on Pd/nFe nanoparticles proceeds byadsorption of the halogenated pollutant on the surface of the bimetallicnanoparticle and subsequent electrophilic substitution of the C\Clbond by atomic H* generated on the Pd surface (hydrodechlorination)(Bokare et al., 2010). The presence of Pd (0.5 wt.%) forms a galvaniccell through which a continuous flow of electrons takes place.Unpalladized iron nanoparticles were not able to degrade 2,3,7,8-TeCDD even at a longer reaction time. Doping of a second metal likePd not only inhibits surface passivation of the iron nanoparticles, butalso contributes to hydrogen gas collection formed in iron corrosion(Nagpal et al., 2010). Disassociation of hydrogen gas on the surface ofthe dopedmetal (Pd) can formmetal hydrides, which are strong reduc-ing agents (Nagpal et al., 2010). Hence, the bimetallic nanoparticleshave the combined property of both (iron and palladium) whichmakes them the catalyst of choice for the removal of such hard-to-degrade compounds.

3.2. Biodegradation of DD: the hybrid treatment

Reductive dechlorination of toxic TeCDD generates monochloro‐dioxins (MCDDs) or DD as the dead end product. Since, DD cannotbe degraded further by Pd/nFe, an alternate process is needed forthe complete mineralization and transformation of DD into nontoxicform. Biodegradation using dioxin degrading bacteria is a promisingmethod for the total eradication of dioxins (Chang, 2008). Therefore,in this study, we used Sphingomonas wittichii RW1 for the degrada-tion of DD, generated from 2,4,7,8-TeCDD, in a sequential treatmentprocess. Sphingomonas wittichii (DSM No: 6014 type Strain, Gene Ac-cession No: X72723, designated as RW1 according to Yabuuchi et al.,2001) is isolated from water samples and maintained in cultivationconditions using medium 457 with dibenzofuran at 30 °C. This strainis known to effectively mineralize DD and DF (Wittich et al., 1992;Wilkes et al., 1996; Hong et al., 2002; Nam et al., 2006). Within 4 h,DDwas completely degraded and stoichiometric generation of catechol

Fig. 1. Dechlorination of TeCDD by Pd/nFe into dibenzodioxin ([TeCDD]0=27 nmol,[Pd/nFe]=0.1 g and pHi=2.5).

was observed which was also subsequently degraded within 18 h(Fig. 2). Whole cell protein quantification revealed that there was nodifference between the control and DD degradation samples, which in-dicated that the amount of the substrate is not sufficient to support bac-terial growth (data not shown).

Since the concentration of DD generated from 2,3,7,8-TeCDDwas very small, bacterial growth and biodegradation studies wereconducted with equal concentrations of DF and MCDD. DF degrada-tion was much faster than DD degradation (DF was completely de-graded within 1 h), which could be attributed to the presence of aDF-activated enzyme system. Unlike DD and DF, the degradationof MCDD was slow, and it took 9 h for 50% degradation and 18 hfor complete degradation (Fig. 3). The biodegradation study evi-dently indicates that not only DD but also MCDD, which can beformed by the reductive cleavage of 2,3,7,8-TeCDD, can be effective-ly degraded by the dioxin degrading bacteria S. wittichii RW1 in a se-quential treatment process.

4. Conclusion

To the best of our knowledge, this is the first report on rapid, effi-cient and absolute degradation of 2,3,7,8-TeCDD under ambient condi-tions (experiments were conducted under mild conditions without theuse of harsh chemicals, heat or pressure) using a nano-bioredox hybridtechnology. After the successful development of the nano-bioprocess

Fig. 3. Depletion ofMCDDand formation ofMCDDdegradation intermediates by resting cellsof DF-grown Sphingomonas wittichii RW1 (0.01–0.02 OD600 at 30 °C, [MCDD]0=27 nmoland pHi=7).

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for the treatment of 2,3,7,8-TeCDD in an aqueous phase, our futurework is focused on testing the feasibility of this process for soilremediation. Since, TeCDD contamination is mostly observed insoils and sediments, ex-situ methodologies like soil mixing/blendingor catalyst spraying technologies will be explored.

Acknowledgment

This work was supported by National Research Foundation ofKorea (NRF) (MEST) (No. 2011‐0028723) and the “The GAIA Project”.

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