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16 Biotransformation and Biodegradation of Organophosphates and Organohalides Ram Chandra and Vineet Kumar CONTENTS 16.1 Introduction......................................................................................................................... 476 16.2 Biotransformation............................................................................................................... 476 16.2.1 Mammalian Biotransformation............................................................................ 477 16.2.1.1 Phase I........................................................................................................ 477 16.2.1.2 Phase II...................................................................................................... 479 16.2.2 Microbial Biotransformation................................................................................. 479 16.2.2.1 Biotransformation of Lindane................................................................480 16.2.2.2 Biotransformation of DDT...................................................................... 481 16.3 Biodegradation....................................................................................................................484 16.3.1 Organophosphates.................................................................................................. 485 16.3.1.1 Fate in Environment and Mode of Action of Toxicity........................ 488 16.3.1.2 Biodegradation of OPs............................................................................. 492 16.3.1.3 Enzymatic Mechanism for Detoxification of OPs............................... 496 16.3.2 Organohalides.........................................................................................................500 16.3.2.1 Alkyl Halides............................................................................................ 501 16.3.2.2 Alkenyl Halides........................................................................................ 501 16.3.2.3 Aryl Halides.............................................................................................. 501 16.3.2.4 Fate in Environment and Mode of Action of Toxicity of Organohalides...................................................................................... 502 16.3.2.5 Biodegradation of Organohalides.......................................................... 503 16.3.2.6 Enzymatic Mechanism for Detoxification of Organohalides............ 512 16.4 Other Processes of Transformation of Pollutants in Environment.............................. 514 16.4.1 Bioremediation........................................................................................................ 514 16.4.2 Biosparging.............................................................................................................. 515 16.4.3 Bioventing................................................................................................................ 516 16.4.4 Biopiling................................................................................................................... 516 16.4.5 Biostimulation......................................................................................................... 516 16.4.6 Bioaugmentation..................................................................................................... 517 16.5 Challenges............................................................................................................................ 517 References...................................................................................................................................... 518

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16.1 Introduction Organophosphorus compounds or organophosphates (OPs) form a large group of chemicals that are most widely used around the world for protecting agricultural crops from insects, pests and weeds, for livestock, human health and as warfare nerve agents. They are also used as plasticizers, stabilizers in lubricating and hydraulic oils, flame retardants, and gasoline additives. One of the main distribution routes for OPs into the environment are believed to be wastewater and discharges from wastewater treatment plants (WWTP). The excessive use of OPs have generated a number of environmental problems such as contamination of air, water and terrestrial ecosystems, harmful effects on different biota, and the disruption of biogeochemical cycling. It is believed that poisoning by between 750,000 and 3,000,000 OPs occur globally every year. OPs act as acetylcholinesterase inhibitors resulting in an accumulation of acetylcholine and the continued stimulation of acetylcholine receptors. Therefore, they are also called anticholinesterase agents. Besides this, organohalides are compounds that contain one or more halogen atom. Organohalides of natural and anthropogenic origin are ubiquitous in the environment. Over 1500 organohalides are known to be produced naturally (Ballschmiter, 2003). Synthetic organohalides have found uses as solvents, refrigerants, insecticides, degreasing agents, pesticides, pharmaceuticals, plasticizer polymers, and medicines. Organohalides are also produced as by products in various industrial processes. The majority of these compounds are chlorinated. However, their uncontrolled release into the environment has caused environmental damage, as many (xenobiotic) organohalides are not only toxic, but also highly recalcitrant to biodegradation, and they readily accumulate in lipids leading to bioaccumulation, often posing the greatest health risk to humans. Chemical and physical methods of decontamination of OPs and organohalides are not only expensive and time consuming, but also in most cases do not provide a complete solution. The elimination of a wide range of OPs and organohalides from the environment is an absolute requirement to promote the sustainable development of our society with low environmental impact. Interest in the microbial biodegradation of these pollutants has intensified in recent years as mankind strives to find sustainable ways to clean up contaminated environments. This biodegradation and biotransformation methods endeavor to harness the astonishing, naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of organic compounds. This chapter focusses on biodegradation and biotransformation of OPs and organohalides through microorganisms in the environment, with an emphasis on how microbial enzymes play a major role in the degradation and detoxification of environmental pollutants. This book chapter explores global challenges on the issues of OPs and organohalides and their fate in the environment.

16.2 Biotransformation Biotransformation is defined as a biological process whereby a substance is changed from one chemical to another (transformed). It involves simple, chemically defined reactions catalyzed by enzymes present in the cells (i.e., microorganisms, plants, and animals). Biotransformation processes are often preferred to chemical processes when high specificity is required, to attack specific sites on the substrate and for a single isomer of the

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product. However, the biotransformation process modifies not only the physicochemical properties of compounds, such as solubility or bioavailability, but also the toxicity level of the given xenobiotics. The terms biotransformation and metabolism (metabolic transformation) are often used synonymously; particularly when applied to drugs. The term metabolism is often used to describe the total fate of a xenobiotic which includes absorption, distribution, biotransformation, and excretion. Metabolism is commonly used to mean biotransformation from the standpoint that the products of xenobiotic biotransformation are called metabolites. There are two major types of biotransformations: (i) xenobiotic biotransformation and (ii) biosynthetically directed biotransformations. In xenobiotic biotransformations, the substrate is foreign to the biological system. Biosynthetically directed biotransformations can be also used to reveal features of the biosynthesis. Biotransformation of xenobiotics occurs in two forms: (i) mammalian biotransformation and (ii) microbial biotransformation. 16.2.1  Mammalian Biotransformation In mammals, biotransformation of xenobiotics and other foreign chemicals catalyzed by enzymes are widely distributed throughout the body. However, the liver is the primary biotransforming organ due to its large size and high concentration of biotransforming enzymes. The kidneys and lungs are next with 10%–30% of the liver’s capacity. A low capacity exists in the skin, intestines, testes, and placenta. Since the liver is the primary site for biotransformation, it is also potentially quite vulnerable to the toxic action of a xenobiotic that is activated to a more toxic compound in the liver and to a lesser extent also in the lung and intestine. An important consequence of mammalian biotransformation is that the physical properties of a xenobiotic are generally changed from those favoring absorption (lipophilicity) to those favoring excretion in urine or feces (hydrophilicity). Without biotransformation, lipophilic xenobiotics would be excreted from the body so slowly that they would eventually overwhelm and kill an organism. However, some chemicals stimulate the synthesis of enzymes involved in xenobiotic biotransformation. This process, known as enzyme induction, is an adaptive and reversible response to xenobiotic exposure. Enzyme induction enables some xenobiotics to accelerate their own biotransformation and elimination. The structure (i.e., amino acid sequence) of a given biotransforming enzyme may differ among individuals, which can give rise to differences in the rates of xenobiotic biotransformation. The reactions catalyzed by xenobiotic biotransforming enzymes is shown in Table 16.1 (Williams, 1971). There are many different processes that can occur and the pathways of drug metabolism can be divided into two phases: phase I and phase II. 16.2.1.1  Phase I Phase I reactions involve hydrolysis, reduction, and oxidation of xenobiotics, as shown in Table 16.1. These reactions expose or introduce a functional group (–OH, –NH2, –SH, or –COOH) and usually result in only a small increase in the hydrophilicity of xenobiotics. In general, phase I biotransformation is often required for subsequent phase II biotransformation as shown in Figure 16.1. Although several enzyme systems participate in phase I metabolism of xenobiotics, perhaps the most notable enzyme in the xenobiotic transformation pathway is catalyzed by cytochrome P450s (CYPs; P450s). The highest concentration of P450 enzymes involved in xenobiotic biotransformation is found in liver endoplasmic reticulum (microsomes), but

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TABLE 16.1 General Pathways of Xenobiotic Biotransformation and Their Major Subcellular Locations Reaction Phase I Hydrolysis

Localization

Oxidation                

Esterase Peptidase Epoxide hydrolase Azo- and nitro-reduction Carbonyl reduction Disulfide reduction Sulfoxide reduction Quinone reduction Reductive dehalogenation Alcohol dehydrogenase Aldehyde dehydrogenase Aldehyde oxidase Xanthine oxidase Monoamine oxidase Diamine oxidase Prostaglandin H synthase Flavin-monooxygenases Cytochrome P450

Microsomes, cytosol, lysosomes, blood Blood, lysosomes Microsomes, cytosol Microflora, microsomes, cytosol Cytosol, blood, microsomes Cytosol Cytosol Cytosol, microsomes Microsomes Cytosol Mitochondria, cytosol Cytosol Cytosol Mitochondria Cytosol Microsomes Microsomes Microsomes

Phase II            

Glucuronide conjugation Sulfate conjugation Glutathione conjugation Amino acid conjugation Acylation Methylation

Microsomes Cytosol Cytosol, microsomes Mitochondria, microsomes Mitochondria, cytosol Cytosol, microsomes, blood

Reduction

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Enzyme

P450 enzymes are present in virtually all tissues. All P450 enzymes are heme-­containing proteins. The heme iron in cytochrome P450 is usually in the ferric (Fe3+) state. When reduced to the ferrous (Fe2+) state, cytochrome P450 can bind ligands such as O2 and carbon monoxide (CO). The CYPs detoxify and/or bioactivate a vast number of xenobiotic chemicals and conduct functionalization reactions that include N- and O-dealkylation,

Xenobiotics

Phase I

Phase II

Exposed or add functional group

Biosynthetic conjugation

Oxidation reduction hydrolysis

Primary product

Lipophilic FIGURE 16.1 Biotransformation of xenobiotic in mammalian system.

Secondary product

Excretion Hydrophilic (ionizable)

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aliphatic and aromatic hydroxylation, N- and S-oxidation, and deamination. The basic reaction catalyzed by cytochrome P450 is monooxygenation in which one atom of oxygen is incorporated into a substrate, designated RH, and the other is reduced to water with reducing equivalents derived from NADPH, as follows:

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Substrate (RH) + O 2 + NADPH + H + Product (ROH) + H 2O + NADP+

Cytochrome P450 catalyzes several types of oxidation reactions, such as hydroxylation of an aliphatic or aromatic carbon, epoxidation of a double bond, heteroatom (S-, N-, and I-), oxygenation and N-hydroxylation, heteroatom (O-, S-, N-, and Si-) dealkylation, oxidative group transfer, cleavage of esters, and dehydrogenation. 16.2.1.2  Phase II Phase II biotransformation is catalyzed often by the “transferase” enzymes that ­perform conjugating reactions. Included in the phase II reaction schemes are glucuronidation, sulfonation (more commonly called sulfation), methylation, acetylation, glutathione ­conjugation, and amino acid conjugation (such as glycine, taurine, and glutamic acid). The products of phase II conjugations are typically more hydrophilic than the parent compounds and therefore usually more readily excretable. Phase II biotransformation of xenobiotics may or may not be preceded by phase I biotransformation. For example, morphine, heroin, and codeine are all converted to morphine-3-glucuronide. In the case of morphine, this metabolite forms by direct conjugation with glucuronic acid. In the other two cases, however, conjugation with glucuronic acid is preceded by phase I biotransformation: hydrolysis (deacetylation) in the case of heroin and O-demethylation (involving oxidation by cytochrome P450) in the case of codeine. Similarly, acetaminophen can be glucuronidated and sulfated directly, whereas phenacetin must undergo phase I metabolism (involving O-deethylation to acetaminophen) prior to undergoing phase II biotransformation. Specific families of phase II xenobiotic-metabolizing enzymes include the UDPglucuronosyltransferases (UGTs), sulfotransferases (STs), N-acetyltransferases (arylamine N-acetytransferase; NATs), glutathione S-transferases (GSTs), and various methyltransferases, such as thiopurine S-methyl transferase and catechol O-methyl transferase. The GSTs function as cytosolic dimeric isoenzymes of 45–55 kDa size that have been assigned to at least four classes: alpha, mu, pi, theta, and zeta; humans possess > 20 distinct GST family members. The conjugation of certain xenobiotics with glutathione is catalyzed by all class of GSTs. For example, the alpha, mu, and pi classes of human GSTs all catalyze the conjugation of 1-chloro-2,4-dinitrobenzene. In concert with the phase I enzymatic machinery, the phase II enzymes metabolize, detoxify, and at times bioactivate xenobiotic substrates in coordination. 16.2.2  Microbial Biotransformation Microbial transformation may be defined as when the transformation of organic compounds occurs by microorganisms. The microorganisms have the ability to chemical modify a wide variety of organic compounds. These microorganisms during the biotransformation process synthesize a wide range of enzymes which act on organic compounds and convert them into other compounds or modify them. Biotransformation processes are mediated by two group of microorganisms especially bacteria and fungi.

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16.2.2.1  Biotransformation of Lindane Hexachlorocyclohexane (HCH), formerly referred to as gamma benzene hexachloride (BHC), exists in several isomeric forms, including alpha (α), beta (β), gamma (γ), delta (δ), Zeta (ζ), eta (η), and theta (θ). The gamma isomer is commonly known as lindane [gammahexachlorocyclohexane (γ-HCH)], as shown in Figure 16.2. The Dutch scientist Dr Teunis van der Linden discovered its insecticidal properties (Hardie, 1964). It can be used as an insecticide and has been used to kill soil-dwelling and plant-eating insects. Lindane and other HCH isomers are highly chlorinated hydrocarbons. The presence of a large number of electron withdrawing chlorine groups makes some of the HCH isomers rather recalcitrant in an oxic environment. The other isomers can be formed during the synthesis of lindane, and have been used either as fungicides or to synthesize other chemicals. In 2005, the production and agricultural use of lindane was banned under the Stockholm convention on persistent organic pollutants (POPs) (Hanson, 2005). The use of lindane had been banned in more than 50 countries and restricted in 33 countries (Humphreys et al., 2008). Further, in 2006, the US EPA cancelled agricultural uses of lindane (ATSDR, 2005). Lindane has a half-life of about two weeks in soil and water. Once in the soil, lindane adsorbs strongly to organic matter and is therefore relatively immobile in the soil. Lindane in soil with especially low organic matter content or subject to high rainfall can leach into surface and even ground soil microflora and aquatic microflora are adversely affected by lindane. Lindane significantly reduced the growth and activity of nitrifying and denitrifying bacteria in soil (Martinez-Toledo et al., 1993; Sáez et al., 2006). In general, the HCH isomers can be biodegraded to a series of less chlorinated organic compounds under both aerobic and anaerobic conditions, and in some cases, HCHs can be used as the sole carbon source for bacterial growth (Rubinos et al., 2007). During the microbial degradation, the chloride atoms of HCHs, which are usually considered to be toxic and xenobiotic, may most commonly be replaced by hydrogen or hydroxyl groups. The efficiency of its microbial degradation in the environment depends on certain biotic and abiotic factors such as the availability of HCH degrading microbes, temperature, pH, moisture, texture and organic content of soil, etc. 16.2.2.1.1  Aerobic Degradation The aerobic degradation pathway of lindane has been studied in some detail for Sphingobium japonicum UT26 (formerly Sphingobium paucimobilis SS86) that was able to use lindane as the sole source of carbon and energy and was isolated from an experimental field to which lindane had been applied (Nagata et al., 1999, 2007). Other HCH-degrading Sphigobium strains, such as S. indicum B90 (Kumari et al., 2002), S. indicum B90A (Dogra et al., 2004) from India and S. francense Sp+ (Ceremonie et al., 2006) from France were also characterized. Lindane is degraded under both aerobic and anaerobic conditions, but it is generally mineralized only under aerobic conditions (Phillips et al., 2005). The aerobic Cl Cl

Cl Cl

Cl Cl FIGURE 16.2 Chemical structure of lindane (γ-HCH).

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degradation of lindane is divided into two pathways: (i) upstream pathway and (ii) downstream pathway. 16.2.2.1.1.1  Upstream pathway  In this pathway two initial dehydrochlorination reactions produce the putative product 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,3,4,6-TCDN) via the observed intermediate γ-pentachlorocyclohexene (γ-PCCH). Subsequently 2,5-dichloro2,5-cyclohexadiene-1,4-diol (2,5-DDOL) is generated by two rounds of hydrolytic dechlorinations via a second putative metabolite, 2,4,5-trichloro- 2,5-cyclohexadiene-1-ol (2,4,5-DNOL). 2,5-DDOL is then converted by a dehydrogenation reaction to 2,5-dichlorohydroquinone (2,5-DCHQ). The major upstream pathway reactions described above are enzymatically catalyzed by LinA (dehydrochlorinase), LinB (halidohydrolase), and LinC (dehydrogenase) proteins, but two other, minor products, 1,2,4-trichlorobenzene (1,2,4TCB) and 2,5-dichlorophenol (2,5-DCP), are produced, presumptively by spontaneous dehydrochlorinations of the two putative metabolites, 1,3,4,6-TCDN and 2,4,5-DNOL. Both 1,2,4-TCB and 2,5-DCP appear to be dead-end products that are not degraded by UT26. 16.2.2.1.1.2 Downstream pathway  Downstream degradation pathway is a reductive dechlorination of 2,5-DCHQ to chlorohydroquinone (CHQ) which is catalyzed by LinD (reductive dechlorinase) protein. The pathway then bifurcates, with the minor route being a further reductive dechlorination by LinD protein to produce hydroquinone (HQ), which is then ring cleaved to γ-hydroxymuconic semialdehyde (γ-HMSA). The conversion of HQ γ-HMSA is catalyzed by LinE (ring cleavage dioxygenase). The major route involves the direct ring cleavage of CHQ to an acylchloride by LinE, which is further transformed to maleylacetate (MA) through the action of LinF (reductase). MA is onverted to β-ketoadipate and then to succinyl coenzyme A (CoA) and acetyl-CoA, which are both metabolized in the tricarboxylic acid (TCA). The complete aerobic degradation pathway of lindane (γ-HCH) is shown in Figure 16.3. 16.2.2.1.1.3  Anaerobic Degradation  Several bacterial species such as Clostridium sphenoides, Clostridium rectum and several other representatives of Bacillaceae and Enterobacteriaceae actively metabolize lindane under anaerobic conditions (Heritage and Mac Rae, 1977; Haider, 1979; Ohisa et al., 1980). The degradation of lindane is initiated with a dechlorination to form pentachlorocyclohexane (PCCH), from which the 1,2-di-chlorobenzene (DCB) and 1,3,-di-chlorobenzene (DCB) isomers and finally mono-chlorobenzene (CB) are formed as shown in Figure 16.4 (Quintero et al., 2005). In the degradation of lindane, intermediate metabolites such as tetrachlorocyclohexene (TCCH) and tetrachlorocyclohexenol (TCCOL) have been detected. During the degradation of lindane by Xanthomonas sp. ICH12, formation of two intermediates, γ-2,3,4,5,6-pentachlorocyclohexene (γ-PCCH) and 2,5-dichlorobenzoquinone (2,5-DCBQ), were identified by gas chromatography-mass spectrometric (GC–MS) analysis (Quintero et al., 2005). γ-PCCH and and 2,5-dichlorohydroquinone were a novel metabolites from HCH degradation (Manickam et al., 2007). 16.2.2.2  Biotransformation of DDT 1,1,1-Trichloro-2,2-di(p-chlorophenyl)-ethane (DDT) is a very important persistent organochlorine pesticide and widely used to control agricultural pests and vectors of malaria, plague, dengue and other insect-borne disease since the 1940s (Wong et al., 2005). The contamination of DDT is of great concern due to its long half-life, recalcitrance to degradation, bioaccumulation and biomagnifications in food chains, and potential toxicity to humans

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2,6-dichlorohydroquinone (2,6-DCHQ) Cl

Succinate

Cl Cl

Cl

Cl Cl

γ-Hexachlorocyclohexane (γ-HCH) LinA Upstream pathway

Cl

Cl

Cl

Cl Pentachlorocyclohexene (γ-PCCH)

Cl O2

LinEb LinE

NAD+ +HCl

HCl Cl

COOH HO

COOH

HO

LinF

2-Chloromaleylacetate (2-CMA)

Cl

Cl

Cl

Spontaneous

HO

HO

Cl

LinE LinEb

HO NADH+H+ NAD+

LinC (LinX)

LinD

C

CoA

COOH C

CoA

Succinyl-CoA

?

H2O

γ-Hydroxymuconic semialdehyde

COOH C=O Cl

Cl

H3C

Downstream pathway O

HCl

Chlorohydroquinone (CHQ) OH

Acetyl-CoA O

LinJ CoA

+

COOH

O2

CO-CoA

O

NAD+

Acylchloride

1,3,4,6,-Tetrachloro-1,4-cyclohexadiene H2O (1,4-TCDN) HCl Spontaneous LinB OH Cl Cl Cl HCl Cl Cl Cl 2,4,5-Trichloro-2,5-cyclohexadiene-1-diol 1,2,4-Trichlorobenzene (2,4,5-DNOL) H2O (1,2,4-TCB) HCl Spontaneous Dead end LinB OH Cl Cl OH HCl Cl 2,5-Dichlorophenol (2,5-DCP)

LinGH

NADH+H Maleylacetate COOH

NADH+H+

β-Ketoadipate (3-oxoadipate), COOH

COOH COOH

LinF

HCl

Cl

Dead end

O

H2O

Spontaneous

HCl

Cl

LinA

HO

Succinyl-CoA β-Ketoadipate (3-oxoadipate),

HO GS-SG+HCl 2GSH

LinD

O2

COOH CHO LinE

Hydroquinone (HQ) OH

TCA cycle

HO

GS-SG+HCl

2GSH 2,5-Dichlorohydroquinone (2,5-DCHQ) Cl

OH

HO

Cl

2,5-Dichloro-2,5-cyclohexadiene1,4-diol (2,5-DDOL)

FIGURE 16.3 Aerobic degradation pathways of lindane (γ-HCH) in Sphingobium japonicum UT26. (Adapted from Nagata Y et al. 2007. Applied Microbiology and Biotechnology 76: 741–752.)

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OH Cl

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Biotransformation and Biodegradation of Organophosphates and Organohalides

Cl Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl 1,2-DCB

Cl γ-

Cl

Cl

Cl

γ-HCH

CB

PCCH

Cl

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1,3-DCB FIGURE 16.4 Degradation routes for lindane (γ-HCH) in Xanthomonas sp. ICH12 under anaerobic conditions.

and wildlife. Its production and use have been banned or restricted in many several countries in the 1970s. Since DDT residues are lipophilic, they tend to accumulate in the fatty tissues of the ingesting organisms along the food chain. DDT is a colorless crystalline substance which is nearly insoluble in water but highly soluble in fats and most organic solvents. Banned for agricultural use worldwide by the 2001 Stockholm Convention on Persistent Organic Pollutants, the use of DDT is still permitted in small quantities in countries that need it, with support mobilized for the transition to safer and more effective alternatives. DDT residues in water and soil are of concern as their uptake can lead to the accumulation of primary products. Their removal from water and soil is therefore a priority. DDT residues have been shown in Figure 16.5 to persist in the environment predominantly in the form of DDT; 1,1,1-trichloro-2-o-chlorophenyl-2-p-chloro-phenylethane (o,p-DDT); 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (o,p-DDD); 1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene (p,p-DDE). DDD and DDE are the transformation products of DDT formed due to microbial action or due to chemical or photochemical reactions. The chemical structure of DDT contains chlorinated aliphatic and aromatic structures that impart great chemical stability. The toxicity of DDT is mainly due to its chlorine atoms, and dechlorination may reduce its environmental risk and biotoxicity. The chemical structure of DDT is shown in Figure 16.5. Microorganisms play an important role in the fate of DDT in natural and controlled environments. A range of bacteria and white rot fungi, such as Eubacterium limosum (Yim et al., 2008), Alcaligenes eutrophus A5 (Nadeau et al., 1994), Boletus edulis (Huang et al., 2007), Fusarium solani (Mitra et  al., 2001), and Phanerochaete chrysosporium (Bumpus and Aust, 1987) have been demonstrated to degrade DDT in both pure culture and natural soil. DDT can be biodegraded by two distinct microbial processes, aerobic oxidative degradation and anaerobic reductive dechlorination. Cl

Cl

Cl

Cl

Cl

Cl

Cl

H

H

H

C

C

C

C

C

C

Cl

Cl

p,p’-DDT FIGURE 16.5 DDT and its residues.

Cl

Cl

Cl

o,p’-DDT

Cl

H

Cl

Cl C

Cl

p,p’-DDD

Cl

C

Cl

p,p’-DDE

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(a)

Cl

(b)

Cl

(c)

Cl

(d)

Cl

(e) Cl

HC

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Cl

CCl3

HO H H HO

HC

CCl3

HC HO HO

Cl

Cl

CCl3

HC O HOOC HO

CCl3 COOH

Cl

FIGURE 16.6 Biotransformation of DDT under aerobic condition by Alcaligenes eutrophus A5. (a) DDT, (b) 2,3-dihydrodiolDDT, (c) 2,3-dihydroxy-DDT, (d) ring fission product, and (e) 4-chlorobenzoic acid.

16.2.2.2.1  Aerobic Transformation The aerobic transformation pathway of DDT by Alcaligenes eutrophus A5 has been studied in detail as shown in Figure 16.6 (Nadeau et al., 1994). A. eutrophus A5 initially oxidizes DDT (compound a) at the ortho and meta positions to form a 2,3-dihydrodiol-DDT intermediate (compound b). It is proposed that this is a dioxygenase type of attack resulting in the transient production of a DDT dihydrodiol (compound b). The dihydrodiol compound is unstable and easily dehydrates into two hydroxylated compounds under weak acidic conditions (pH