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International Biodeterioration & Biodegradation 65 (2011) 921e930

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International Biodeterioration & Biodegradation

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Review

Basic studies and applications on bioremediation of DDT: A review

Adi Setyo Purnomoa, Toshio Morib, Ichiro Kameic, Ryuichiro Kondob,*aDepartment of Chemistry, Faculty of Mathematics and Natural Sciences, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya 60111, IndonesiabDepartment of Agro-Environmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japanc Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, Miyazaki 889-2192, Japan

a r t i c l e i n f o

Article history:Received 7 June 2011Received in revised form12 July 2011Accepted 12 July 2011Available online 16 September 2011

Keywords:BioremediationDegradationDDTBrown-rot fungiCompostSpent mushroom waste

* Corresponding author. Tel./fax: þ81 92 642 2811.E-mail addresses: adi.spurnomo@yahoo.com, adi_s

0964-8305/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.ibiod.2011.07.011

a b s t r a c t

The persistent insecticide DDT (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane) has been widely used forpest control in the management of mosquito-borne malaria and is still used for that purpose in sometropical countries. Considering the potential for negative effects due to DDT contamination, it is neces-sary to determine effective methods of remediation. Several methods have been used to degrade ortransform DDT into less toxic compounds. Bacteria and white-rot fungi (WRF) have been shown toenhance the degradation process in soil using both pure and mixed cultures. Recently, a biologicalapproach has been used as an environmentally-friendly treatment, using new biological sources todegrade DDT, e.g. brown-rot fungi (BRF), cattle manure compost (CMC) and spent mushroom waste(SMW). In this review, the abilities of BRF, CMC and SMW to degrade DDT are discussed, including themechanisms and degradation pathways. Furthermore, application of these sources to contaminated soilis also described. The review discusses which is the best source for bioremediation of DDT.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9222. Degradation of DDT by brown-rot fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 922

2.1. Identification of metabolic products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9222.2. Involvement of the Fenton reaction in DDT degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9222.3. Degradation of DDT and its metabolites by the chemical Fenton reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9232.4. Mechanisms of involvement of the Fenton reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9232.5. DDT degradation pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924

3. DDT degradation potential of cattle manure compost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9243.1. DDT degradation by cattle manure compost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9243.2. Isolation and identification of fungi from cattle manure compost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9253.3. DDT degrading assay on isolated fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 925

4. Degradation of DDT by mushroom waste medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9254.1. Degradation and mineralization of DDT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9264.2. Ligninoliytic enzymes activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926

5. Application on contaminated soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9265.1. Application of brown-rot fungi in artificially DDT-contaminated soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9265.2. Degradation of artificially DDT-contaminated soil by cattle manure compost and its isolated fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9275.3. Degradation of artificially DDT-contaminated soil by spent mushroom waste of Pleurotus ostreatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9275.4. Degradation of DDT in historically contaminated soil by spent mushroom waste of Pleurotus ostreatus . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 928

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928

etyo@chem.its.ac.id (A.S. Purnomo), ryukondo@agr.kyushu-u.ac.jp (R. Kondo).

All rights reserved.

A.S. Purnomo et al. / International Biodeterioration & Biodegradation 65 (2011) 921e930922

1. Introduction have ligninolytic enzymes, it has been proposed that they usehydroxyl radicals produced via the Fenton reaction for degradation

Table 1DDT degradation ability of brown-rot fungi in pure culture (Purnomo et al. 2008,2010c).

Brown-rotfungi

DDT decrease(%)

Metabolites (%)

DDE DDD DBP DBHa DDMUa

G. trabeum 61.8� 1.4 30.1� 1.5 22.6� 3.8 13.9� 0.8 þ �F. pinicola 63.1� 4.2 � 61.8� 3.1 � � �D. dickinsii 46.8� 4.2 24.1� 3.9 19.9� 3.1 � � þ

Initial concentration of DDT is 0.25 mmol. Data are presented as mean� standard devi-ations (n¼ 3). DDT (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane); DDE (1,1-dichloro-2,2-bis (4-chlorophenyl) ethylene);DDD (1,1-dichloro-2,2-bis (4-chlorophenyl) ethane);DBP (4,4-dichlorobenzophenone); DBH (4,4-dichlorobenzhydrol); DDMU (1-chloro-2,2-bis (4-chlorophenyl) ethylene).

a The data were determined by GC/MS: (�) undetectable; (þ) detectable.

DDT (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane) was intro-duced as an insecticide during World War II to combat mosquitoeswhich spread malaria and typhus (Busvine 1989; Boul 1995; Foghtet al. 2001). DDT was the first synthetic insecticide and gainedpopularity all over the world. However, its use has recently beenprohibited inmostcountriesbecauseof itsnegative impactonwildlifeand its ill effects on human health via the food web (Kale et al. 1999;Foghtet al. 2001). Lowdirectexposure toDDT leads to symptomssuchas headache, nausea, vomiting, confusion, and tremors. Furthermore,accumulation of DDT in the body can affect the nervous system,increase tumor production, and is associated with pancreatic cancer(Turusov et al. 2002; Gautam and Suresh 2007). The presence ofchlorine atoms in DDT and its metabolites, in conjunction with theirlow solubility and tendency to partition preferentially into the lipo-philic phase, makes them highly ecotoxic, especially to higherorganisms. However, DDT is still being used in some developingcountries for essential public health purposes (Foght et al. 2001). TheUnited States Environmental Protection Agency (EPA) has classifiedDDT and its metabolite products, DDD (1,1-dichloro-2,2-bis (4-chlorophenyl) ethane) and DDE (1,1-dichloro-2,2-bis (4-chlorophenyl) ethylene), as priority pollutants (Sayles et al. 1997;Foght et al. 2001). Considering the potential for negative effects, it isnecessary to address theenvironmental persistenceof this insecticideand to find effective methods of remediation.

The removal of DDT from contaminated soils has become anenvironmental priority, and both physicochemical and biologicalremediation processes have been studied. Although chemical andphysical treatments are more rapid than biological treatments, theyare generally more destructive and intrusive to affected soils, moreenergy intensive, and often more expensive than bioremediation(Foght et al. 2001). Considering the bioremediation options, severalbacteria and white-rot fungi (WRF) have been shown to enhancedegradation processes in soil, using both pure and mixed cultures(Guenzi and Beard 1967; Wedemeyer 1967; Subba-Rao and Alex-ander 1985; Bumpus and Aust 1987; Fernando et al. 1989; Bumpuset al. 1993; Xu et al. 1994; Boul 1996; Hay and Focht 2000; Kama-navalli and Ninnekar 2004; Thomas and Gohil 2011; Xie et al. 2011).Recently, bioremediation of DDT was performed using anenvironmentally-friendly biological approach by means of somenew biological sources to degrade DDT, including brown-rot fungi(BRF, Purnomo et al. 2008, 2010a, 2011), cattle manure compost(CMC, Purnomo et al. 2010b) and spent mushroom waste (SMW,Purnomo et al. 2010c). BRF including Gloeophyllum trabeum, Fomi-topsis pinicola and Daedalea dickinsii have shown a good ability todegrade DDT, with the Fenton reaction being an important part ofthe system (Purnomo et al. 2008, 2010a, 2011, Villa et al. 2008).Mucor circinelloides andGalactomyces geotrichumwere isolated fromCMC, which is good at degrading DDT in artificially contaminatedsoil (Purnomo et al. 2010b). In addition, mineralization of DDT bySMWof Pleurotus ostreatus in artificially contaminated soil has beenreported, and proposed as a promising source of DDT bioremedia-tion (Purnomo et al. 2010c). Previously, there have been two reviewsonmicrobial degradation of DDT: one by Aislabie et al. (1997) whichcovers literature up to 1997 about microbial degradation of DDT andits residues, and the other by Foght et al. (2001) which coversliterature up to 2001 about bioremediation of DDT-contaminatedsoil. In this article, we have updated this knowledge by reviewingrecent works on the bioremediation of DDT.

2. Degradation of DDT by brown-rot fungi

Unlike the process using bacteria and WRF, DDT biodegradationby BRF has received relatively little attention. Because BRF do not

of various compounds (Purnomo et al. 2008, 2010a). In BRF,extracellular Fenton-type mechanisms have been reported to beinvolved in the degradation of several xenobiotic compounds(Wetzstein et al. 1997; Kerem et al. 1999; Wetzstein et al. 1999;Schlosser et al. 2000; Jensen et al. 2001; Newcombe et al. 2002).Twelve species of BRF were screened for their ability to degradeDDT in which G. trabeum, F. pinicola and D. dickinsii showed thegreatest abilities to degrade DDT (Purnomo et al. 2008). In thissection, characterization of the major metabolic products, clarifi-cation of the degradation pathways, and an examination of the roleof the Fenton reaction is discussed in detail.

2.1. Identification of metabolic products

The metabolic products were identified by HPLC and GC/MS onthe basis of the retention time and absorption maximum at specificwavelengths in comparison with standards (Table 1). DDE, DDD,DBP (4,4-dichlorobenzophenone) and DBH (4,40-dichlorobenzhy-drol) were detected as metabolic products of DDT degradation byG. trabeum. However, when D. dickinsii was used the metabolicproducts were DDE, DDD and DDMU (1-chloro-2,2-bis (4-chlorophenyl) ethylene), while only DDD was detected whenF. pinicolawas used. These results show that the different fungi havedifferent transformation pathways of DDT (Purnomo et al. 2008,2010a).

The mineralization of [14C]DDT by G. trabeum, F. pinicola andD. dickinsii was evaluated. Less than 0.25% of the initial [14C]DDT(378 kdpm) was mineralized to [14C]CO2. Because these minerali-zation rates are very low, it is unlikely that the fungi can mineralizeDDT (Purnomo et al. 2008, 2010a). Bumpus and Aust (1987)reported that the mineralization of [14C]DDT by G. trabeumwas< 0.1% in both nitrogen-deficient and nitrogen-sufficientcultures. Thus, it appears that BRF cannot mineralize DDT. In thecase of the DDT degradation pathway in the WRF P. chrysosporium,DBP appears to be an end-intermediate compound, which issubsequently converted by “unknown” ring cleavage processes toproduce CO2 (Bumpus and Aust 1987).

2.2. Involvement of the Fenton reaction in DDT degradation

DDT degradation by G. trabeum was enhanced by an iron-dependent reaction, resulting in the production of different meta-bolic products. DBP was not detected as a metabolic product of DDTin themediumwithout FeSO4, indicating that DBPwas produced bythe iron-dependent reaction (Purnomo et al. 2008). For F. pinicola,where DDD was detected as a metabolic product, degradation ofDDTwas not enhanced by an iron-dependent reaction. Degradation

A.S. Purnomo et al. / International Biodeterioration & Biodegradation 65 (2011) 921e930 923

of DDT by D. dickinsii also did not require an iron-dependentreaction (Purnomo et al. 2010a).

For further evidence of the involvement of the Fenton reaction,the concentration of hydroxyl radicals was determined. F. pinicolaand D. dickinsii produced lower concentrations of hydroxyl radicalscompared with G. trabeum (Purnomo et al. 2008, 2010a). Further-more, the presence of mannitol as a hydroxyl radical scavengerinhibited DDT degradation by G. trabeum but not by D. dickinsii andF. pinicola. Since D. dickinsii and F. pinicola produced low amounts ofhydroxyl radicals, this suggests that the production of hydroxylradicals alone is not enough to degrade DDT (Purnomo et al. 2010a).For F. pinicola, addition of Fe2þ to the culture caused the productionof hydroxyl radicals to increase. However, DDT degradation andDDD production decreased. Since an increase in hydroxyl radicalscaused a decrease in DDT degradation and DDD production, thissuggests that hydroxyl radicals might inhibit the ability of fungus todegrade DDT. F. pinicola might be sensitive to hydroxyl radicalssince the radicals are known to attack many biomolecules due totheir high reactivity. Besides, increasing of Fe2þ concentrationenhanced DDT degradation by D. dickinsii due to an increase in thenumber of hydroxyl radicals. The results indicate that if therequired amount of hydroxyl radical is reached, D. dickinsii candegrade DDT via the Fenton reaction (Purnomo et al. 2010a). Asimilar tendency was seen in G. trabeum. The added Fe2þ might beoxidized rapidly to yield Fe3þ as the predominant form of iron inthe cultures. G. trabeum can reduce Fe3þ and thus drive the Fentonreaction extracellularly (Jensen et al. 2001). Furthermore, anextracellular metabolite; 2,5-dimethoxy-1,4-benzoquinone(DMBQ) was detected from G. trabeum as a key component forthe Fenton reaction cycle. This finding was supported by Keremet al. (1999), who identified DMBQ as an important component ofextracellular oxidative systems in G. trabeum to operate a redoxcycle. The fungal mycelium reduced DMBQ to 2,5-dimethoxy-1,4-hydroquinone (DMHQ). DMHQ reduces Fe3þ to Fe2þ and producesextracellular H2O2 via a nonenzymatic reaction. This pathwayprovides evidence that G. trabeum uses a benzoquinone-drivenFenton reaction to produce the hydroxyl radical (Kerem et al.1999). However, DMBQ was not detected in the F. pinicola andD. dickinsii cultures. This demonstrates that these fungi produce

Fig. 1. Transformation of DDT (R¼ Cl) or DDD (R¼H) or DDE (R

a low level of hydroxyl radicals due to their lack of a Fenton reactioncycle system. Supplemented Fe2þ could be used directly to stimu-late the Fenton reaction causing oxidation to Fe3þ. However, sincethe fungi lack a redox system, the reaction will not occur for long(Purnomo et al. 2010a).

Different levels of hydroxyl radical production in cultures mayalso be caused by different amounts of H2O2 production by fungi.G. trabeum produced about 50e70 mM of H2O2 (Purnomo et al.2008). However, lower amounts of H2O2 were detected fromF. pinicola (16 mM) and D. dickinsii (21 mM) (Purnomo et al. 2010a).Since G. trabeum has a Fenton reaction cycle system, the higheramount of hydroxyl radical production in G. trabeum is caused bythe higher amount of H2O2. These findings support the suppositionthat a benzoquinone-driven Fenton reaction cycle is important forG. trabeum to degrade DDT.

2.3. Degradation of DDT and its metabolites by the chemical Fentonreaction

To prove that DBP was produced by the Fenton reaction, DDTand its metabolites (DDE, DDD and DBP) were treated via thechemical Fenton reaction (CFR) in the samemanner as in the fungalculture (70 mM H2O2 and 100 mM FeSO4). DDT, DDE and DDD(0.25 mmol) were degraded approximately 32%, 34%, and 44%,respectively. DBP was detected as the main metabolic productapproximately 20%, 31%, and 37%, respectively (Purnomo et al.2008). These results indicate that a Fenton reaction is possiblyinvolved in the DDT degradation pathway, particularly in thetransformations of DDT, DDE and DDD to DBP. In application,approximately 75% of initial DDT in slurry soil (1.6 mg g�1) wasdegraded by CFR (Villa et al. 2008).

2.4. Mechanisms of involvement of the Fenton reaction

A mechanism of the direct transformations of DDT, DDE andDDD to DBP was proposed (Fig. 1). For DDT, the benzylic hydrogenlocated between the two rings is expected to be easily attacked bythe hydroxyl radical, yielding a resonance-stabilized, carbon-centered benzylic radical. This radical will react with oxygen,

¼OH) to DBP by the Fenton reaction (Purnomo et al. 2008).

A.S. Purnomo et al. / International Biodeterioration & Biodegradation 65 (2011) 921e930924

yielding a peroxyl radical, which can then attack hydrogen from thesurroundings to yield the peroxide. In the presence of Fe2þ, theperoxide will be reduced to an alkoxyl radical, which will undergobeta scission to yield the benzophenone (DBP) and trichloromethylradical (CCl3). The same pathway could likely operate in the case ofDDD, yielding DBP and dichloromethyl radical (CHCl2) (Purnomoet al. 2008). For DDE, the transformation mechanism involvingthe hydroxyl radical is based on the anti-Markovnikov rule. Thehydroxyl radical is assumed to be electron-deficient and electro-philic. The alkene double bond is attacked by the hydroxyl radical,yielding a resonance-stabilized, carbon-centered benzylic radical.This radical will react with oxygen, yielding a peroxyl radical, whichcan then attack hydrogen from the surroundings to yield theperoxide. In the presence of Fe2þ, the peroxidewill be reduced to analkoxyl radical, which will undergo beta scission to yield benzo-phenone (DBP) and a dichlorohydroxymethyl radical (COHCl2,Purnomo et al. 2008).

2.5. DDT degradation pathway

On the basis of the identification of the metabolic products, theDDT degradation pathway in BRF is proposed as shown in Fig. 2.G. trabeum initially dehydrochlorinates DDT to form DDE, followedby hydrogenation to DDD. DDD then undergoes oxidativedechlorination to DBP, followed by reduction to DBH (Purnomoet al. 2008, 2010a). This pathway differs from the proposedpathways in bacteria and other fungi described above, particularlyin the transformation of DDE to DDD (Purnomo et al. 2008). Aswith G. trabeum, D. dickinsii also initially dehydrochlorinates DDTto form DDE, followed by hydrogenation to DDD, and thenundergoes dechlorination to DDMU. This pathway differs from theproposed pathway in G. trabeum described above, particularly inthe transformation of DDD to DDMU. F. pinicola dechlorinates DDTto DDD where DDD is the end product. These results indicate thatDDT degradation pathways differ between BRF (Purnomo et al.2010a).

DDT, DDD and DDE may also be directly transformed to DBP viathe involvement of a Fenton reaction (Purnomo et al. 2008, 2010a).A benzoquinone-driven Fenton reaction cycle is a key to BRFdegradation of DDT. Because BRF do not have lignin-degradingenzymes, it is also possible that BRF use an enzymatic systemthat differs from that of WRF.

Fig. 2. Proposed DDT degradation pathway of brown-rot fungi. Thin white, open, and dottedThick black arrows show transformation by Fenton reaction. DDT (1,1,1-trichloro-2,2-bis (4-cdichloro-2,2-bis (4-chlorophenyl) ethane); DBP (4,4-dichlorobenzophenone); DBH (4,4-dich2008, 2010a).

3. DDT degradation potential of cattle manure compost

Since composts are rich sources of xenobiotic-degradingmicroorganisms, they can be used to degrade pollutants or totransform pollutants into less toxic substances. Composts arereportedly capable of degrading a variety of organic contaminants(Laine and Jorgensen 1996; Semple and Fermor 1997; Semple et al.1998; Eggen 1999; Reid et al. 2002; Lau et al. 2003; Puglisi et al.2007). However, DDT biodegradation by composts has receivedrelatively little attention (Kantachote et al. 2003; Lourencetti et al.2007). In this section, the ability of cattle manure compost (CMC) todegrade DDT is discussed. A detailed investigation into degradationprocesses and the biodegradation ability of each composting stage(mesophilic, thermophilic, and maturation) was performed. DDT-degrading fungi were isolated, identified, and tested for theirability to degrade DDT.

3.1. DDT degradation by cattle manure compost

Under optimal conditions, composting proceeds throughthree stages: (1) the mesophilic stage, which occurs for a fewdays; (2) the thermophilic stage, which can last from a few daysto several months; and (3) the maturation stage, which lasts forseveral months (Tuomela et al. 2000). Cattle manure compost(CMC) was made from cattle manure, rice bran, and pearlite.After mixing, some of the composting material was taken andused as the mesophilic stage material. The remaining compostmaterial was placed into a reactor for composting, with airsupplied at a rate of 0.5 Lmin�1. Once the temperature increased,some of the compost material was taken from the reactor andused as the thermophilic stage material. After the temperaturedropped and remained at a relatively constant level, the compostmaterial was taken and used as maturation stage material(Purnomo et al. 2010b).

During composting, the CMC passed from the mesophilic to thethermophilic stage after 36 h. The maximum temperature occurredat 9 h and then steadily dropped. After 120 h, the temperaturestabilized, indicating the maturation stage. During the initial 45 h,the mesophilic and thermophilic stages were critical points in theDDT degradation process when thermophilic microbes degradeDDTmore effectively than those present during the mesophilic andmaturation stages (Purnomo et al. 2010b). At the beginning of

arrows indicate transformation by G. trabeum, D. dickinsii and F. pinicola, respectively.hlorophenyl) ethane); DDE (1,1-dichloro-2,2-bis (4-chlorophenyl) ethylene); DDD (1,1-lorobenzhydrol); DDMU (1-chloro-2,2-bis (4-chlorophenyl) ethylene) (Purnomo et al.

Table 2DDT degradation ability of isolated fungi from cattle manure compost at differenttemperature (Purnomo et al. 2010b).

Strain no. Name of species Degradation rate (%)

30 �C 60 �C

Mesophilic1CMC1 Galactomyces geotrichum 83.6� 3.4aA 90.1� 2.9aA1CMC2 Mucor circinelloides 81.9� 7.2aA 72.9� 1.2bB1CMC3 Mucor circinelloides 85.9� 2.2aA 81.9� 5.2aA1CMC4 Mucor circinelloides 82.1� 3.7aA 79.6� 3.2aA1CMC5 Mucor circinelloides 89.4� 2.7aA 55.4� 5.9bB1CMC6 Mucor circinelloides 95.3� 1.4aA 88.7� 4.1bB1CMC7 Mucor circinelloides 89.9� 1.5aA 80.4� 3.4aB1CMC8 Mucor circinelloides 88.5� 8.2aA 82.9� 4.2aA

Thermophilic2CMC1 Galactomyces geotrichum 85.1� 2.1aA 91.9� 1.1aB2CMC2 Galactomyces geotrichum 80.4� 3.9aA 93.5� 2.6aB

Maturation3CMC1 Mucor circinelloides 84.5� 1.2aA 69.8� 1.4bB3CMC2 Mucor circinelloides 81.9� 5.2aA 75.1� 4.8aA

Initial concentration of DDT is 0.25 mmol. Data are presented as mean� standarddeviations (n¼ 3). Data followed by the same minor letter on each column or by thesame capital letter on each row are not statistically different from each other(P< 0.05).

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composting, mesophilic bacteria predominate, but after thetemperature increases to over 40 �C, thermophilic bacteria andfungi also appear in the compost. When the temperature exceeds60 �C, microbial activity decreases dramatically, but after thecompost has cooled, mesophilic bacteria and actinomycetes againdominate (McKinley and Vestal 1985; Strom 1985).

To investigate the process of DDT degradation by CMC indetail, the ability of the materials at each stage of the CMCprocess was investigated. The experiments were conducted at 30or 60 �C, temperatures that are representative of mesophilic/maturation or thermophilic conditions, respectively. The ther-mophilic stage material demonstrated the highest degradationability, indicating that thermophilic stage materials degraded DDTmore effectively than materials at the other stages. The ability ofthermophilic microbes to degrade DDT may be more effectivethan those present during the mesophilic/maturation stages(Purnomo et al. 2010b). In the composting environment, ther-mophilic microbes are an important biodegradation agent, evenin small populations (McKinley and Vestal 1985; Strom 1985;Tuomela et al. 2000). The results provide direct evidence thatduring the composting process, DDT was degraded to a greaterextent during the thermophilic stage, with DDD was detected asa major metabolic product (Baczynski et al. 2010). However, sinceDDD production was not equivalent to DDT degradation, it islikely that some other metabolic by-products were produced. It ispossible that DDD could be transformed into other metabolicproducts such as DBP (Baczynski et al. 2010; Purnomo et al.2010b).

The mineralization of [14C]DDT (378 kdpm) by materials at eachstage of the CMC at 30 and 60 �Cwas also studied. The thermophilicstage showed the highest mineralization ability at 60 �C. The resultsalso provide direct evidence that during the composting process,DDT was mineralized to a greater extent during the thermophilicstage (Kantachote et al. 2003; Purnomo et al. 2010b). However,because the mineralization rate is very low (less than 1.00%), it isassumed that CMC could not mineralize DDT (Purnomo et al.2010b).

3.2. Isolation and identification of fungi from cattle manurecompost

Fungi play a very important role in the biodegradation andconversion processes during composting. In addition, fungi usemany carbon sources and can survive in extreme conditions, andare mainly responsible for compost maturation (Sparling et al.1982; McKinley and Vestal 1985; Strom 1985; Wiegant 1992;Tuomela et al. 2000). However, several bacteria has been isolatedand identified from contaminated soils such as Staphylococcus(Songkong et al. 2008), Basillus, Clostridium, Escherichia (Purnomoet al. 2010b), and Alcaligenes (Xie et al. 2011). Bacteria were moresensitive to DDT than actinomycetes and fungi. DDT-resistantbacterial strains showed more promise in degrading DDT thanisolated fungal strains (Kantachote et al. 2003).

Several fungi were isolated from CMC, which most of the fungiisolated from the mesophilic and maturation stages were 99e100%identical at the nucleotide level to Mucor circinelloides (Table 2). M.circinelloides is one of the most commonly occurring fungi of theMucor genus (Purnomo et al. 2010b). Most of the fungi belonging tothe Mucor genus have been reported to possess the ability todegrade xenobiotic compounds (Anderson et al. 1970; Andersonand Lichtenstein 1971, 1972; Shetty et al. 2000; Su et al. 2006;Szewczyk and D1ugo�nski 2009). In addition, all isolated fungifrom the thermophilic stage were 100% identical with Galactomycesgeotrichum (Table 2) (Purnomo et al. 2010b). This fungus has beenreported to degrade dyes (Jadhav et al. 2008a,b).

3.3. DDT degrading assay on isolated fungi

The ability of isolated fungi to degrade DDT in PDB medium at30 or 60 �C was determined. The fungi isolated from materials atthe thermophilic stage (G. geotrichum) exhibited a significantlyhigher degradation ability at 60 �C (Table 2) (Purnomo et al. 2010b).This indicated that thermophilic fungi have greater activity ata higher temperature although they have adequate activity ata lower temperature. Most of the fungi isolated from materials atthe mesophilic and maturation stages (M. circinelloides) demon-strated a greater ability to degrade DDT at 30 �C (Table 2). Theseresults indicate that even though they have activity during thethermophilic stages, a lower temperature is more suitable for theiractivity. This finding demonstrate that most of the fungi isolatedfrom CMC materials possess the ability to degrade DDT at 30 and60 �C. Approximately 16% and 6% of DDD and DBP were detected asmetabolic products, respectively (Purnomo et al. 2010b). Based onidentification of metabolic products, DDT was initially reductivelydechlorinated to formDDD, followed by oxidative dechlorination toDBP. The processes involve single electron transfer, removal ofa chlorine ion, and formation of an alkyl radical (Archer 1973; Foghtet al. 2001). In addition, soil fungus Fusarium solani had ability todegrade DDT and its metabolites such as DDD, DDE, DDOH and DBP(Mitra et al. 2001).

4. Degradation of DDT by mushroom waste medium

There have been several studies of DDT degradation by WRF inliquid cultures, in which the process has been assumed to bemediated by the fungal lignin-degrading system (Wedemeyer1967; Bumpus and Aust 1987; Fernando et al. 1989; Bumpus et al.1993; Boul 1996; Hay and Focht 2000; Kamanavalli and Ninnekar2004; Chung et al. 2009; Purnomo et al. 2010c, Xiao et al. 2011).WRF species produce different enzymes depending on their geneticand growth conditions. Key degradation enzymes include ligninperoxidase (LiP), manganese peroxidase (MnP), versatile peroxi-dase (VP) and laccase. The lignin and xenobiotic degradationpotential of these enzymes arewell documented (Lamar 1992; Vyaset al. 1994; Zhao and Yi 2010). However, bioremediation by fungi inliquid cultures requires costly preparations prior to application,

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especially for the medium. To evaluate the effectiveness of biore-mediation of DDT-contaminated soil, several parameters must beinvestigated first, and the degradation ability might be reduced incontaminated soil compared with liquid cultures (Purnomo et al.2010c). In mushroom production, 5 kg of spent mushroom waste(SMW) is generated from the production of every kilogram ofmushrooms (Semple et al. 2001). High levels of residual nutrientsand enzymes are still present in SMW. When using WRF forbioremediation, the availability of fungal inocula is a practicalconsideration and the use of SMW could be advantageous due tothe low cost and environmentally-friendly treatment (Purnomoet al. 2010c). SMWs have been well documented in their degrada-tion of some xenobiotics (Semple et al. 1998; Eggen 1999; Law et al.2003; Puglisi et al. 2007; Chung et al. 2009).

4.1. Degradation and mineralization of DDT

Since high levels of residual nutrients and enzymes remain inSMW, it might be used advantageously as a low-cost bioremedia-tion tool to degrade DDT. Various cultivated fungal inocula werescreened for their ability to degrade DDT. Fungal substrate fromPleurotus ostreatus showed the highest ability to degrade DDT(Chung et al. 2009; Purnomo et al. 2010c). Since>25% of worldwidemushroom production is Pleurotus mushrooms, it would beadvantageous to use Pleurotus SMW. Thus, the fungal substrategenerated before mushroom production and SMW generated aftermushroom production from P. ostreatus were used in the detailedinvestigation. SMW was significantly better at degrading DDT thanthe fungal substrate. Eggen (1999) has reported that degradation ofPAHs by SMW from P. ostreatus was higher than that by the fungalsubstrate before mushroom production.

Mineralization of [U-14C]DDT was also investigated. [U-14C]DDT(378 kdpm) was mineralized by 4.4% and 5.1% during the 56d incubation by the P. ostreatus fungal substrate and SMW,respectively (Purnomo et al. 2010c). Bumpus and Aust (1987)reported that [U-14C]DDT was 6% mineralized during a 30 d incu-bation at room temperature in a low nitrogen liquid culture ofP. ostreatus. This suggests that the mineralization of [U-14C]DDT insolid and liquid matrices is not very different. Based on thedegradation and mineralization results, the degradation of DDT byP. ostreatus SMW is more effective than by the fungal substratebefore production (Purnomo et al. 2010c).

4.2. Ligninoliytic enzymes activities

Bioremediation activity is dependent on the ability of the fungalspecies colonized in the substrate to produce oxidative ligninolyticenzymes such as laccase, VP, MnP, and LiP. These enzymes areresponsible for the degradation of lignin as well as of pollutants(Buswell et al. 1993; Rodríguez et al. 2004). Therefore, the lig-ninolytic enzyme activities of P. ostreatus fungal substrate and SMWwere determined. Among the ligninolytic enzymes, the MnPactivity showed high activities for both the fungal substrate andSMW (Purnomo et al. 2010c). The degradation of some pollutantsbyWRF has beenwell correlatedwith the ligninolytic activity of thefungi (Bumpus and Aust 1987). LiP andMnP had an influence on theefficiency of DDT degradation (Chung et al. 2009). It is also possiblethat VP may be involved in DDT degradation. It has been reportedthat VP can oxidize phenolic and non-phenolic aromaticcompounds as well as Mn2þ to Mn3þ and that it act as a diffusibleoxidizing agent (Rodríguez et al. 2004). However, since the lig-ninolytic enzyme activities in the SMWwere significantly differentthan in the fungal substrate, but the mineralization rate was notsubstantially different, the results indicate that the higher miner-alization rate of DDT by P. ostreatus SMW was not caused by the

higher ligninolytic enzyme activities. However, the high MnPactivity in both fungal substrate and SMW suggests that MnPmightbe involved in DDT degradation even though it is not the mainmechanism (Purnomo et al. 2010c). Further investigation into DDTdegradation by directly ligninolytic enzymes is needed.

It is also possible that intracellular enzymes may be involved inDDT degradation. It has been reported that cytochrome P450monooxygenase (P450) is involved in the degradation of somepollutants. DDT is metabolized under aerobic conditions by theP450 enzyme to DDD, dicofol, FW-152, 2,2-bis(4-chlorophenyl)acetic acid (DDA) and DBH (Purnomo et al. 2010c; Suhara et al. inpress). It can be assumed that the intracellular P450 also has animportant role in the metabolism of persistent aromaticcompounds, alongside ligninolytic enzymes.

5. Application on contaminated soil

Soils are a complex environment containing mixed populationsof microorganisms with synergistic and antagonistic activities. Soilis not an inert matrix, and its properties and the prevailing envi-ronmental conditions will influence the behavior of both microor-ganisms and DDT (Deepthi et al. 2007; Mwangi et al. 2010).Therefore, it is important to examine DDT degradation in soil, bothin the laboratory under controlled conditions and in the field(Guenzi and Beard 1967; Xu et al. 1994; Boul 1996). Firstly, the DDTdegradation abilities of BRF, CMC and SMW of P. ostreatus wereinvestigated in artificially contaminated soils in the laboratoryunder controlled conditions. The best source was selected andapplied on historically contaminated soil.

5.1. Application of brown-rot fungi in artificiallyDDT-contaminated soil

G. trabeum, F. pinicola and D. dickinsii all displayed a good abilityto degrade DDT in pure culture (Purnomo et al. 2008, 2010a). In thissection, the ability of these fungi to degrade DDT in soil wasinvestigated to find out whether these fungi are suitable forbioremediation purposes. DDT was degraded by G. trabeum,F. pinicola and D. dickinsii in both sterilized (SL) and un-sterilized(USL) soils (Table 3). Considering the DDT degradation rates inboth SL and USL soils, G. trabeum has the best ability to degrade DDTin artificially contaminated soil (Purnomo et al. 2011). Bycomparing the DDT degradation rates in SL and USL soils, thedegradation of DDT by G. trabeum in USL soil was higher than in SLsoil (Table 3), indicating that soil microorganisms enhanced theability of G. trabeum to degrade DDT. However, the degradation ofDDT by F. pinicola in USL soil was lower than in SL soil (Table 3),indicating that soil microorganisms may inhibit the ability ofF. pinicola to degrade DDT. Finally, the degradation of DDT byD. dickinsii in USL and SL soils did not show any significant differ-ence, which indicates that soil microorganisms did not inhibit orenhance the ability of this fungus to degrade DDT (Table 3,Purnomo et al. 2011). In the USL soil, DDD was detected asa metabolic product. Because DDD was not detected in the SL soil,the production of DDD might be related to the involvement of soilmicroorganisms. Some soil bacteria have been reported to trans-form DDT to DDD via reductive dechlorination (Katayama et al.1993; Megharaj et al. 2000).

The use of Fenton reaction system for the treatment ofcontaminated soils was initially investigated by Watts et al. (1990),who was the first to observe pentachlorophenol mineralization.Subsequently, several other works appeared which emphasized theefficiency of Fenton processes for the remediation of soilscontaminated with other organic pollutants (Watts et al. 1994; Villaand Nogueira 2006; Villa et al. 2008). As the Fenton reaction

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Fig. 3. Degradation and mineralization of DDT in contaminated soil by G. trabeum,CMC and P. ostreatus SMW during 28 d incubation period. Bars represent degradationrate and triangles represent mineralization rate. Data points represent means andstandard deviations (n¼ 3).

Table 3DDT degradation rates in artificially contaminated sterilized (SL) and un-sterilized (USL) soils by brown-rot fungi during a 14 d incubation period (Purnomo et al. 2011).

Brown-rot fungi DDT decreased (%)a

Sterilized soil Un-sterilized soil

Without addition Fe2þ With addition Fe2þ Without addition Fe2þ With addition Fe2þ

G. trabeum 36.2� 1.8aA 40.8� 0.9bA 41.6� 1.0aA 42.8� 0.8aAF. pinicola 8.8� 1.0aB 8.7� 0.2aB 4.3� 1.1aB 28.7� 3.7bBD. dickinsii 11.5� 1.0aC 15.1� 0.4bC 11.0� 0.4aC 32.3� 0.7bC

Initial concentration of DDT is 0.25 mmol. The data were determined by HPLC. Data are presented as mean� standard deviations (n¼ 3). Data followed by the same lower caseletter on each row or by the same capital letter in each column are not significantly different (P< 0.05). DDT (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane).

a vs. Control.

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enhanced DDT degradation by BRF in pure cultures (Purnomo et al.2008, 2010a), the effect of the addition of Fe2þ as a source for theFenton reaction in DDT contaminated soil was also investigated.Although the soil system may already contain iron, the concen-tration of iron may not be high enough to assist in degradation ofDDT because of its use in other reactions, especially for F. pinicolaand D. dickinsii. The Fe concentrations in soil found differed ina range from few mM Fe to about 200 mM Fe (Ammari and Mengel2006). The supplement of Fe2þ (10 mM) was expected to enrichthe soil and also enhance DDT degradation. In comparison to theDDT degradationwithout the addition of Fe2þ, G. trabeum degradedDDT better with the addition of Fe2þ than without it both SL andUSL soils. Similarly, D. dickinsii and F. pinicola also degraded DDTbetter with the addition of Fe2þ than without it (Table 3, Purnomoet al. 2011). In the USL soil, DDD was produced by G. trabeum,F. pinicola and D. dickinsii. The addition of Fe2þ to F. pinicola andD. dickinsii enhanced DDT degradation significantly, causing anincrease in DDD production (Purnomo et al. 2010a, 2011).

5.2. Degradation of artificially DDT-contaminated soil by cattlemanure compost and its isolated fungi

The greatest amount of degradation occurred by mesophilicstage material in both SL and USL soils (Purnomo et al. 2010b).Higher degradation in the USL soil was caused by the activities ofsoil microorganisms. Since active soil microbes alone cannotdegrade DDT, the synergistic actions between compost and soilmicroorganisms might enhance DDT degradation (Purnomo et al.2010b). Three isolated fungi from each stage of the compostingprocess exhibited an ability to degrade DDT at 30 �C:M. circinelloides 1CMC6, G. geotrichum 2CMC1, and M. circinelloides3CMC1. These fungi were selected to examine degradation of DDTin artificially contaminated soil. The fungi degraded DDT(0.25 mmol) by approximately 50% and 85% in SL and USL soils,respectively (Purnomo et al. 2010b). These results indicate that soilmicroorganisms enhanced the abilities of these fungi to degradeDDT.

In the USL soil, DDD and DBP were detected as metabolicproducts. It seemed that DDD, which is produced by soil microor-ganisms, was transformed into DBP by the fungi (Purnomo et al.2010b). This would suggest that the fungi prefer to degrade DDDover DDT. Approximately 27% and 5% of DDD and DBP were alsodetected as metabolic products in the SL soil, respectively. Theisolated fungi and soil microorganisms possess a comparable abilityto transform DDT into DDD. However, since the amount of DDD inthe USL soil was higher than in the SL soil, it appears that soilmicroorganisms produce DDD to a greater extent than the fungi,with the transformation of DDT to DDD by soil microorganismsdominating (Kantachote et al. 2003, 2004; Lourencetti et al. 2007;Purnomo et al. 2010b). Based on these results, isolated fungidemonstrated a high ability to degrade DDT in both SL and USL

soils, showing that isolated fungi can be used for the bioremedia-tion of DDT contamination in soil. This supports the evidence thatCMC contains fungi which have the ability to degrade DDT in soil.

5.3. Degradation of artificially DDT-contaminated soil by spentmushroom waste of Pleurotus ostreatus

P. ostreatus fungal humus showed the ability to degrade DDT(0.25 mmol) in the soil with an efficiency of 50e70% after 30d (Purnomo et al. 2010c). Furthermore, DDT was degraded by about40% and 80% by P. ostreatus SMW in SL and USL soils, respectively.DDD was detected as the major metabolic product in the USL soil.Since DDD was not detected in the SL soil, the production of DDDmight be caused by the involvement of soil microorganisms(Purnomo et al. 2010c). The mineralization of [U-14C]DDT in arti-ficially contaminated soil was also investigated. During the 56d incubation, [U-14C] DDT (378 kdpm) was mineralized by 5.1% and8.0% by P. ostreatus SMW in SL and USL soils, respectively. Severalstudies have found that mineralization of DDT in soils is typicallyvery low (<3% after a 42 d incubation (Nair et al. 1992; Boul 1996)or longer (Guenzi and Beard 1968; Zayed et al. 1994)). Mineraliza-tion activity in the control was 1.1%, which indicated mineralizationby soil microorganisms (Purnomo et al. 2010c). This result providesfurther evidence for the potential role of soil microbes to enhanceDDT degradation (Deepthi et al. 2007).

The distribution of [U-14C] intermediate metabolic productswas investigated. From both SL and USL soils; DDD, DDMU andDDMS (1-chloro-2,2-bis (4-chlorophenyl) ethane) were detectedin the acetone and n-hexane extracts. Additionally, DDA (bis (4-chlorophenyl) acetic acid) was detected as a metabolic productin the water extract. From the CO2 formation results, the lowerlevel of intermediate compounds in the water-fraction of the USLsoil might be due to the increased formation of CO2. This suggests

Fig. 4. Degradation of DDT in historically contaminated soil by spent mushroomwasteof P. ostreatus during 28 d incubation period. Data points represent means and stan-dard deviations (n¼ 3). The same minor letter on each line indicates no statisticaldifference (P< 0.05).

A.S. Purnomo et al. / International Biodeterioration & Biodegradation 65 (2011) 921e930928

that soil microorganisms enhanced the transformation of water-soluble intermediate compounds to CO2 (Purnomo et al. 2010c).However, the possibility of synergistic interactions betweenP. ostreatus SMW and soil microorganisms needs further detailedinvestigation.

5.4. Degradation of DDT in historically contaminated soil by spentmushroom waste of Pleurotus ostreatus

Even though BRF and CMC have a high ability to degrade DDT inartificially contaminated soil, these sources lack mineralizationability (Fig. 3). Since SMW of P. ostreatus has a good ability todegrade and mineralize DDT in artificially contaminated soil(Fig. 3), this source was applied to historically contaminated soilwhich has higher concentrations of DDT. In historically contami-nated soil, the DDT concentration (0.8 mmol) is more than threetimes higher than the DDT concentration in the artificiallycontaminated soil (0.25 mmol). Compared with the control, about70% of DDT was degraded by SMW of P. ostreatus in historicallycontaminated soil (Fig. 4). This means that about 30% of the DDTremained in the soil, at a concentration of 42.5 ppm. This studygives evidence that SMWof P. ostreatus is a potential natural sourcewhich can be used for the bioremediation of DDT in “real world”application.

Further detail investigation on degradation pathway andmechanism is needed to achieve complete mineralization of DDT.Since mineralization rate was still low, developing method isneeded to obtain complete mineralization of DDT. Therefore,some methods are needed to stimulate Hiratake for perfectmineralization of DDT. The use of mixed cultures of some WRFcould be advantageous to gain perfect mineralization rate. SinceWRF have ligninolytic enzymes system to degrade DDT, mixedcultures can be used to enhance mineralization ability. Besides,the use mixed cultures of WRF along with bacteria could beadvantageous to enhance DDT mineralization. Involvement ofbiosurfactant, and effect of co-metabolic compounds will be alsoof interest. Since ring-cleavage bacteria have good ability tocleave DDT, further degradation to CO2 should proceed easily byassociated with WRF.

6. Conclusions

Recent work used a biological approach for an environmentally-friendly treatment using biological sources to degrade DDT andalso applied it for bioremediation in contaminated soil. Some BRFwere screened for their ability to degrade DDT with G. trabeum,

F. pinicola and D. dickinsii proposed as new candidates for DDTbioremediation. In application on artificially contaminated soil,G. trabeum showed the highest ability to degrade DDT in both SLand USL soils. The ability of CMC to degrade DDT was also inves-tigated, with DDT degraded during composting and DDD detectedas a metabolic product. Some fungi were isolated and identifiedfrom CMC, and most of them were closely related toM. circinelloides and G. geotrichum. These fungi demonstrateda high ability to degrade DDT at both 30 and 60 �C in pure cultureand artificially contaminated soil. Furthermore, SMWof P. ostreatushas the ability to degrade and mineralize DDT in pure culture andartificially contaminated soil. Since SMW of P. ostreatus has a highability to degrade and mineralize DDT in contaminated soilcompared with other sources, SMW from P. ostreatus is proposedas the best bioremediation source in DDT-contaminated environ-ments. Mineralization is important in bioremediation, indicatingthat the process of degradation of the pollutant into a non toxiccompound (CO2) is complete. After application to historicallycontaminated soil, about 70% of the initial DDT concentration inthe soil was degraded by SMW of P. ostreatus, indicating that thissource is very suitable for bioremediation of DDT in soil. Furtherresearch in situ and ex situ are needed to promote DDT bioreme-diation as a viable treatment option.

Acknowledgments

This work was supported by a grant from the Research Projectfor Ensuring Food Safety from Farm to Table, Ministry of Agricul-ture, Forestry and Fisheries, Japan (PO-3216).

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