Applied Microbiology and Biotechnology Springer-Verlag 2005 10.

1007/s00253-004-1864-3

Mini-Review

Biodegradation of xenobiotics by anaerobic bacteria

Chunlong Zhang1 and George N. Bennett2 (1) Department of Environmental Sciences, University of Houston-Clear Lake, Houston, TX 77058, USA (2) Department of Biochemistry and Cell Biology, Rice University, 6100 Main St., Houston, TX 77005, USA George N. Bennett Email: gbennett@bioc.rice.edu Phone: +1-713-3484920 Fax: +1-713-3485154 Received: 28 September 2004 Revised: 29 November 2004 Accepted: 30 November 2004 Published online: 26 January 2005 Abstract Xenobiotic biodegradation under anaerobic conditions such as in groundwater, sediment, landfill, sludge digesters and bioreactors has gained increasing attention over the last two decades. This review gives a broad overview of our current understanding of and recent advances in anaerobic biodegradation of five selected groups of xenobiotic compounds (petroleum hydrocarbons and fuel additives, nitroaromatic compounds and explosives, chlorinated aliphatic and aromatic compounds, pesticides, and surfactants). Significant advances have been made toward the isolation of bacterial cultures, elucidation of biochemical mechanisms, and laboratory and field scale applications for xenobiotic removal. For certain highly chlorinated hydrocarbons (e.g., tetrachlorethylene), anaerobic processes cannot be easily substituted with current aerobic processes. For petroleum hydrocarbons, although aerobic processes are generally used, anaerobic biodegradation is significant under certain circumstances (e.g., O2-depleted aquifers, oil spilled in marshes). For persistent compounds including polychlorinated biphenyls, dioxins, and DDT, anaerobic processes are slow for remedial application, but can be a significant long-term avenue for natural attenuation. In some cases, a sequential anaerobic-aerobic strategy is needed for total destruction of xenobiotic compounds. Several points for future research are also presented in this review.

Introduction

Anaerobic biodegradation of xenobiotic compounds has been a subject of extensive research during the last two decades. Consequently, our current understanding of the dissipation mechanisms of xenobiotics in natural anaerobic environments has considerably improved. Many anaerobe-based bioreactors and remediation systems have been developed to effectively clean-up contaminated media. The purpose of this review is to summarize recent advances in our understanding and briefly describe biotechnological applications for the biodegradation of five major groups of xenobiotic compounds: petroleum hydrocarbons and related fuel additives, nitroaromatic compounds and explosives, chlorinated aliphatic and aromatic compounds, pesticides, and surfactants. The review is not intended to be exhaustive, but focuses on representative anaerobes, their biochemical mechanisms, and potential biotechnological and environmental implications. Several excellent reviews have been published on anaerobic biodegradation of xenobiotics, both in general (Janke and Fritsche 1985; Mogensen et al. 2003a; Schink 2002) or focused on specific compounds including petroleum hydrocarbons (Chakraborty and Coates 2004; Heider and Fuchs 1997; Prince 1993; Spormann and Widdel 2000; Widdel and Rabus 2001), explosives (Ahmad and Hughes 2000; Esteve-Nuez et al. 2001; Gorontzy et al. 1994; Marvin-Sikkema and de Bont 1994; Peres and Agathos 2000), chlorinated compounds (Abramowicz 1990; Bedard 2003; Chen 2004; El Fantroussi et al. 1998; Fetzner 1998; Haggblom et al. 2000; Ohtsubo et al. 2004), and pesticides (Sethunathan 1973; Williams 1977). There are several reasons why anaerobic biodegradation of xenobiotics is important to researchers and practitioners. Aerobic processes require expensive O2 delivery systems, maintenance is often high due to biofouling in subsurface remedial applications (Baker and Herson 1994), and there are high energy costs and sludge production when bioreactors are employed (Jewell 1987; McCarty and Smith 1986). In addition, anaerobic conditions naturally prevail in most cases for contaminated groundwater, and some xenobiotic compounds [e.g., tetrachloroethylene, polychlorinated biphenyls (PCBs), and nitro-substituted aromatics] can be efficiently transformed or mineralized only by anaerobic bacteria. In some cases, aerobic degradation does not occur without a prior anaerobic process (Master et al. 2002).

Major groups of anaerobic organisms involved in xenobiotic biodegradation

Like their aerobic counterparts, anaerobic bacteria able to degrade xenobiotic compounds are diverse and present in various anaerobic habitats, including sediments, water-laden soils, gastrointestinal contents, reticulo-ruminal contents, feedlot wastes, sludge digesters, groundwater, and landfill sites (Williams 1977). Anaerobes use natural organics such as proteins, carbohydrates, and many others as carbon and energy sources. Many of the so-called xenobiotic compounds of environmental concern have naturally occurring relatives (Wackett et al. 2002). For other xenobiotics, repeated exposure has resulted in the adaptation and evolution of anaerobic bacteria capable of metabolizing these man-made compounds. Table 1 lists the major groups of anaerobic microorganisms involved in biodegradation of selected xenobiotic compounds. The pure bacterial cultures given in this table are by no means exhaustive

but are representative of each compound category. In reporting these bacteria with compoundspecific metabolic capability, two classical strategies are commonly employed. Some researchers have chosen to employ pure cultures of previously isolated anaerobic strains to test with specific compounds, whereas others have focused on the isolation and identification of new strains from anaerobic bacterial consortia or enrichment cultures (El Fantroussi et al. 1998). Without a systematic screening approach, the number of bacterial cultures successfully isolated is limited since only a small portion of what is present in the actual microbial habitat has been tested. In other cases, several syntrophic bacterial strains of a bacterial consortium co-exist to metabolize a specific compound (El Fantroussi et al. 1998; Janke and Fritsche 1985; Williams 1977). Despite these limitations, the diversity of anaerobic microorganisms able to biodegrade xenobiotic compounds is apparent. Table 1 Major groups of anaerobic microorganisms involved in xenobiotic biodegradation. PAH Polycyclic aromatic hydrocarbon, MTBE methyl tert-butyl ether, TNT trinitrotoluene, DNT dinitrotoluene, RDX hexahydro-1,3,5-trinitro-1,3,5-triazine, HMX octahydro-1,3,5,7-tetranitro1,3,5,7-tetrazocine, PCE tetrachloroethylene, TCE trichloroethene, DCE cis-dichloroethene, VC vinyl chloride, PCB polychlorinated biphenyls, PCP pentachlorophenol, LAS linear alkylbenzene sulfonate, LAEOs linear alcohol ethyoxylates Bacteria namea, source of isolationb, chemical Compounds Reference action Alkane D. oleovorans (P): mineralizes C12C20 n-alkane Aeckersberg et al. 1991 G. spp. (P): oxidizes benzene in Fe(II)-reducing conditions Coates et al. 2001; RooneyBenzene Dechloromonas spp. (S): mineralizes benzene into Varga et al. 1999 CO2 in 5 days G. metallireducens (S): first pure culture (Fe3+ reducing) for toluene oxidation Chakraborty and Coates 2004; Toluene Lovley et al. 1989 Azoarcus and Thauera spp. (S/D): facultative toluene-oxidizing nitrate-reducers Thauera-related (S/P): denitrifying bacteria Ball et al. 1996; Rabus and Ethylbenzene completely mineralize ethylbenzene Widdel 1995 D. acetonicum- and Desulfosarcina variabilisHarms et al. 1999; Hess et al. Xylene related: mineralizes o- and m-xylene 1997; Rabus and Widdel 1995 Acidovorax, Bordetella, Pseudomonas, Sphingomonas, and Variovorax (S): degradation Eriksson et al. 2003; Rockne et PAHs complete for naphthalene and partial for 35 ring al. 2000 PAHs; P. stutzeri and Vibriop pelagius related (S): mineralizes 720% naphthalene Pure aerobes isolated; slow under anaerobic Finneran and Lovley 2001; MTBE conditions, no pure anaerobes isolated Stocking et al. 2000 Veillonella alkalescens (D): the earliest evidence of Esteve-Nuez et. 2001; Hughes anaerobic TNT degradation C. spp. and TNT, DNT et al. 1999; McCormick et al. Desulfovibrio spp. (N): most extensively studied 1976 genera transforming TNT

Compounds

RDX, HMX

PCE, TCE

VC PCBs

PCP

Dioxins Chlorinated pesticide

Bacteria namea, source of isolationb, chemical Reference action Desulfovibrio spp. (S): uses RDX and HMX as sole N source Providencia sp., and M. morganii (S): transforms into nitroso derivatives Boopathy et al. 1998; Kitts et al. Serratia marcescens (M): RDX ring cleavage 1994; Young et al. 1997; Zhang similar to McCormick s pathway and Hughes 2003; Zhao et al. 2002 C. acetobutylicum (N): transforms RDX into NHOH and NH2 derivatives K. pneumoniae (D): degrades RDX into HCHO, CO2 and N2O A. woodii, C. formicoaceticum, Methanolobus El Fantroussi et al. 1998; tindarius, Methanosarcina sp., Methanosarcina Fathepure and Boyd 1988; mazei, Methanosarcina thermphila, Sporomusa Jablonski and Ferry 1992; ovata (N): previously known strains transforming Terzenbach and Blaut 1994 PCE & TCE Desulfitobacterium sp. (S): transforms PCE to TCE and trace amount of DCE Dehalobacter restrictus (S): transforms PCE to De Bruin et al. 1992; Gerritse et ethene al. 1996; Gerritse et al. 1999; Desulfitobacterium frappieri (S/D): tranforms PCE Magnuson et al. 2000; Maymo& TCE into cis-DCE Gatell et al. 1997; Sung et al. Dehalococcoides ethenogenes (N): completes PCE 2003 & TCE degradation into ethene Desulfuromonas michiganensis (S): able to grow on free-phase PCE Dehalococcoides sp. (A): able to grow with VC and He et al. 2003 transform VC into ethene Desulfitobacterium dehalogenans (S): Wiegel et al. 1999 dehalogenates flanking Cl of OH-PCBs Desulfitobacterium frappieri (S/D): 9099% PCP removal forming 3-CP Beaudet et al. 1998; Bouchard Desulfitobacterium halogenans (S), et al. 1996; Shelton and Tiedje Desulfitobacterium chlororespirans (C), 1984; Tartakovsky et al. 1999 Desulfomnile tiedje (N): dechlorinates at o- and mposition Dehalococcoides sp. (S): uses dioxins as the sole Bunge et al. 2003 electron acceptor C. sp. (N): degrades DDT as the sole C source. Ruppe et al. 2003, 2004; Degrades other chlorinated pesticides Sethunathan 1973; Williams

Compounds

P-based pesticide Carbamate pesticide Dinoseb pesticide Anionic surfactant Nonionic surfactant Cationic surfactant
a

Bacteria namea, source of isolationb, chemical action Aerobacter aerogenes, K. pneumoniae, N. vulgaris (S): DDT-degrading Dehalospirilum multivorans: preferentially dechlorinates technical toxaphene Flavobacterium sp. (S): attacks P-insecticides including diazino and parathion K. pneumoniae (D): uses three chlorinated striazines as the sole N source C. bifermentans (D): utilizes Dinoseb as a sole C via cometabolism Strain RZLAS (D): the only pure anaerobe using LAS as the sole S source Pelobacter propionicus & A. sp. (D): LAEOs fermented to CH4 and CO2 Unable to isolate a single bacterium using cationic surfactant as the sole C source

Reference

1977

Sethunathan 1973 Ernst and Rehm 1995; Hammill and Crawford 1996 Denger and Cook 1999 Wagener and Schink 1988 Madsen et al. 2001

Bacteria: A Acetobacterium, C Clostridium, D Desulfobacterium, G Geobacter, K Klebsiella, M Morganella, N Nocardia, P Pseudomonas b Source: A Aquifer materials, C compost; D sludge; M manure; N not specified; P petroleum related sites; S soil or sediment c Chemicals: CP chlorophenol, Dinoseb 2-sec-butyl-4,6-dinitrophenol

Pure cultures summarized in Table 1 have been isolated under strict anaerobic conditions (sulfatereducing and methanogenic). Example bacteria in this category include Clostridia, Desulfobacterium, Desulfovibrio, Methanococcus, Methanosarcina, and most of the newly isolated dehalogenating bacteria (e.g., Dehalococcoides). For practical purposes, some of the facultative denitrifying microorganisms are also included in the table such as Flavobacterium and Klebsiella to illustrate their potential role in these environmental communities. Anaerobic bacteria isolated from environmental compartments and bioreactors are preferentially illustrated over anaerobes of pathological origin. Attention has focused on the isolation of anaerobic bacteria that play a role in the degradation of two types of compounds due to their widespread environmental problems: the petroleum hydrocarbons [benzene-toluene-ethylbenzene-xylene (BTEX); polycyclic aromatic hydrocarbons (PAHs)] and chlorinated compounds including the pesticide DDT [1,1,1-trichloro-2,2-bis(pchlorophenyl)ethane]. In particular, extensive efforts have focused on the latter, partly because halogenated organic compounds probably cause about half of the environmental problems attributable to organic pollution in the world today (Tiedje et al. 1993), and partly because anaerobic biodegradation is a preferred strategy. Following the discovery of the insecticidal properties of DDT in the late 1930s, its subsequent use and the awareness of its environmental persistence, more than 300 bacterial strains have been shown to convert DDT into DDD [1,1dichloro-2,2-bis(p-chlorophenyl)ethane] (Cookson 1995) and several novel dechlorinating strains

have been reported (Chacko et al. 1966; Guenzi and Beard 1967; Matsumura and Boush 1971; Wedemeyer 1966) from the late 1960s to the 1970s. Research on the biodegradation of DDT declined drastically after it was banned in the 1970s (Quensen et al. 2001) and the focus during the last 10 years has been directed toward chlorinated aliphatic hydrocarbons due to their worldwide prevalence. A pure culture of Dehalococcoides ethenogenes was able to completely dechlorinate tetrachloroethylene (PCE) into innocuous ethene (Magnuson et al. 2000; Maymo-Gatell et al. 1997; McCarty 1997), and Desulfuromonas michiganensis can even grow on free-phase PCE (Sung et al. 2003). Most PCE-dechlorinating bacteria convert PCE into trichloroethene (TCE) or further into cis-dichloroethene (DCE) (Bagley and Gossett 1990), while for others the more toxic vinyl chloride (VC) is produced as the end-product. Several recent efforts have therefore been made to isolate VC-transforming bacteria. Dehalococcoides sp., which can grow on VC and transform it into ethene in the presence of lactate and pyruvate as electron donors (He et al. 2003), is one such isolate. Anaerobic degradation of the monoaromatic BTEX hydrocarbons was considered to be negligible prior to the 1980s, partially due to the favorable energetics of aerobic bacteria (Chakraborty and Coates 2004). These compounds have been shown to serve as carbon and energy sources for diverse anaerobic bacteria under nitrate-reducing, Fe(III)-reducing, sulfate-reducing and methanogenic conditions. Except for p-xylene, isolation of pure bacterial cultures degrading all other BTEX compounds has been successful (Table 1). Like BTEX, 2- to 4-ring PAHs are quite readily biodegradable aerobically (Cerniglia 1992), and anaerobic degradation of PAHs was formerly thought impossible. However, naphthalene biodegradation through denitrification has been documented (Eriksson et al. 2003; Mihelcic and Luthy 1988), and phenanthrene biodegradation through similar conditions was also reported (Rockne and Strand 1998). A few PAH-degrading bacterial strains have been successfully isolated but none were able to produce complete mineralization. As a concurrent contaminant with BTEX and PAHs in many petroleumcontaminated sites, methyl tert-butyl ether (MTBE) is mainly susceptible to aerobic degradation; however, anaerobic metabolism of MTBE has been reported (Finneran and Lovley 2001; Kolhatkar et al. 2002; Somsamak et al. 2001; Stocking et al. 2000). Anaerobic degradation of halogenated phenol, particularly pentachlorophenol (PCP), has been the subject of several studies due to its wide use as a wood preservative. Pure cultures able to dechlorinate PCP into 3-chlorophenol have been isolated; some bacteria preferentially remove Cl at the ortho and meta positions (Beaudet et al. 1998; Tartakovsky et al. 1999). However, no single bacterial culture with an ability for complete dechlorination and mineralization has yet been isolated. For polychlorinated biphenyls (PCBs), although reductive dechlorination has been observed frequently in many contaminated sediments and aquifers with an array of microorganisms (Quensen et al. 1988), only recently have pure cultures been characterized (Wu et al. 2002a, b). A strain was isolated that could dechlorinate hydroxylated PCBs (Wiegel et al. 1999). A pure culture that could use dioxin as the sole electron acceptor was isolated (Bunge et al. 2003). The isolation of dioxin-degrading bacteria is a good example of how bacteria have evolved to metabolize toxic xenobiotic compounds. The biodegradation of nitroaromatic explosives [trinitrotoluene (TNT); dinitrotoluene (DNT)] has been studied for more than two decades. Clostridium and Desulfovibrio spp. have been extensively studied for their pathways transforming these compounds into amino- and hydroxyamino-

derivatives under anaerobic conditions. Unlike aerobic mineralization pathways (e.g., DNT mineralization can be readily demonstrated under aerobic conditions, Zhang et al. 2000a, b), significant mineralization of TNT and DNT under anaerobic conditions has never been achieved and anaerobic mineralizing bacteria never isolated. On the other hand, for non-aromatic explosives such as RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (octahyrdo-1,3,5,7-tetranitro1,3,5,7-tetrazocine), pure bacterial cultures able to transform both agents have been isolated (Boopathy et al. 1998; Kitts et al. 1994; Young et al. 1997; Zhao et al. 2002). With a significant number of pesticides in use, dissimilar chemical structures and limited pure bacterial isolates, generalizations regarding pesticide-degrading microorganisms are difficult to make. For instance, in the United States alone, over 125 herbicides, 300 insecticides and 325 fungicides are registered (Cookson 1995). The most extensively studied pesticide has been DDT due to its persistent nature in the environment. The biodegradability of many other new synthetic pesticides are of less concern due to the shorter half-life associated with biotic and abiotic processes. Furthermore, studies on the biodegradation of pesticides appear to be focused mostly on aerobic bacteria, despite some limited studies on the isolation of anoxic bacterial cultures (e.g., Ruppe et al. 2003, 2004). Synthetic surfactants have created environmental problems due to the use of alkyl benzene sulfonate (ABS) detergents that were later replaced by linear alkylbenzene sulfonate (LAS) in the late 1960s. A common misconception is that surfactants are readily removed through aerobic processes in municipal wastewater treatment plants due to sorption and aerobic biodegradation. This is also why biodegradability data of surfactants are predominantly aerobic (Swisher 1987). A significant percentage of surfactants escape aerobic processes and accumulate in anaerobic sludge digesters. A conservative estimate shows that approximately 20% of surfactants reached the anaerobic compartment (AISE and CESIO 1999). In addition, renewed interest in surfactant biodegradation is based on the recent finding that many alkyl phenol polyethoxylates show toxicity to fish and are suspected of being endocrine disrupters. While the importance of anaerobic pathways is still in debate, research efforts to isolate anaerobic surfactant degrading bacteria (Table 1) are limited.

Biochemistry of xenobiotic biodegradation

Hydrocarbons and fuel additives
The anaerobic biochemical pathways of petroleum hydrocarbons and related fuel additives have been the subjects of many investigations during the last two decades. For hydrocarbons, the elucidation of anaerobic BTEX (particularly toluene) degradation pathways is probably the most advanced (Boll et al. 2002). This is not surprising since saturated alkanes are less of a health concern, although quantitatively they are more important than BTEX (Gieg and Suflita 2002). Saturated alkanes are more susceptible to aerobic bacterial attack than unsaturated aliphatic hydrocarbons (i.e., alkene, alkyne). It is also well established that alkanes with long carbon chains and straight structures are more prone to aerobic biodegradation and the same is likely to be the case for anaerobes. The most common aerobic pathway for alkane degradation is oxidation of the terminal methyl group into a carboxylic acid through an alcohol intermediate, and eventually

complete mineralization through -oxidation (Cookson 1995; Leahy and Colwell 1990). Several physiologically and phylogenetically distinct anaerobes have been shown to degrade alkanes (Aeckersberg et al. 1991; Ehrenreich et al. 2000; Rabus et al. 2001; Rueter et al. 1994). Methane can also be formed from alkanes by anaerobic organisms (Zengler et al. 1999). Recent data with an n-hexane-utilizing denitrifying isolate pointed to a pathway involving initial enzymatic attack by fumarate (OOCCH=CHCOO) addition in a manner similar to that for toluene as discussed below (Krieger et al. 2001; Rabus et al. 2001; Wilkes et al. 2002). Another pathway reported in a sulfatereducing bacterium, Hxd3 (Aeckersberg et al. 1991), involves carboxylation followed by removal of a terminal two-carbon unit to reduce the original alkane length by one carbon as the fatty acid is formed (So et al. 2003). Observations of a carbon addition reaction internal to the chain were also made in studies of strain SK-01 (So and Young 1999a, b). Similarly, anaerobic MTBE metabolism is not as well understood as aerobic pathways. In the presence of oxygen, aerobes attack MTBE with a monooxygenase. The biochemical mechanisms of the recalcitrant ether bond cleavage have been explained in a review by Fayolle et al. (2001). With anaerobic bacteria, the cleavage involves methyl transferases and tetrahydrofolate for the degradation of lignin (a naturally occurring ether compound) and hydroxyl group addition during fermentation of polyethylene glycols (-O-CH2-CH2OH). Anaerobic degradation of MTBE has been demonstrated using compound-specific carbon isotope analyses in a groundwater site (Kolhatkar et al. 2002), and transformation of MTBE has been observed under sulfate-reducing conditions (Somsamak et al. 2001). Figure 1 delineates the major enzymes and intermediates involved in anaerobic degradation of BETX compounds. Variations in pathways exist since various bacteria depend on different electron acceptors corresponding to differing redox conditions (Chakraborty and Coates 2004). Complete mineralization has been reported for all BTEX compounds except p-xylene, and research has elucidated the initial enzymatic reactions shown in Fig. 1. A difference from aerobic mechanisms, which involve molecular oxygen, is the introduction of oxygen through H2O to form oxygenated monoaromatic compounds that are susceptible to further ring cleavage. In some cases, for example in the anaerobic degradation of p-cresol, oxidation of the methyl group via addition of oxygen derived from water occurs (Bossert et al. 1989; Bossert and Young 1986). Also shown in Fig. 1 is benzoyl coenzyme A (benzoyl-CoA), a common intermediate for BTEX compounds. BenzoylCoA is formed through the addition of fumarate to the BTEX compounds through the enzymatic action of benzylsuccinate synthase (BSS) (for toluene) or methylbenzylsuccinate synthase (for oand m-xylene) (Biegert et al. 1996). Studies on the mechanism have demonstrated that these are glycyl radical enzymes (Beller and Spormann 1998; Krieger et al. 2001; Leuthner et al. 1998). After formation of benzylsuccinate, it is converted to the CoA derivative benzylsuccinyl-CoA by a CoA transferase and then oxidized to benzoyl-CoA and succinyl-CoA for further metabolism (Leutwein and Heider 1999). The genes encoding the benzyl succinate synthase have been isolated (Hermuth et al. 2002) and, in strain EbN1, are near another operon encoding enzymes required for conversion of benzyl succinate to benzoyl-CoA (Kube et al. 2004). The enzyme benzylsuccinylCoA dehydrogenase is encoded by bbsG in Thauera aromatica (Leutwein and Heider 2002). Benzoyl-CoA is transformed to 1,5-diene-1-carboxyl-CoA through the key enzyme, benzoyl-CoA reductase. After a series of hydration and dehydrogenation steps, 3 mol acetyl-CoA and 1 mol CO2 is formed from each mole of BTEX compound (Mogensen et al. 2003a).

Fig. 1 Anaerobic pathways for the biodegradation of petroleum hydrocarbons [benzene-tolueneethylbenzene-xylene (BTEX); adapted from Chakraborty and Coates 2004; Mogensen et al. 2003]. A Fumarate (HOOCCH=CHCOOH), E1 benzylsuccinate synthase (BSS), E2 ethylbenzylsuccinate synthase, E3 ethylbenzene dehydrogenase, E4 ethylbenzylsuccinate synthase, E5 benzoyl-CoA reductase The anaerobic biochemical pathways for PAHs have been studied only in the last few years, with a focus on naphthalene and phenanthrene. Pure cultures of sulfate-reducing (Galushko et al. 1999) and nitrate-reducing (Rockne et al. 2000) bacteria that degrade naphthalene have been isolated. Like monoaromatic hydrocarbons, research has focused on the rate-limiting step of the initial enzymatic attack. In contrast to earlier work that supported phenol as the major intermediate in the fermentation of naphthalene [D. Grbic-Galic (1990) Microbial degradation of homocyclic and heterocyclic aromatic hydrocarbons under anaerobic conditions. Unpublished report, Department of Civil Engineering, Stanford University], recent work by several research groups has identified

2-naphthoic acid (2-NA) as a common intermediate (Fig. 2) (Zhang et al. 2000a, b). This acid is formed through carboxylation with the addition of a C1 unit (Zhang and Young 1997) or fumarate, catalyzed by naphthyl-2-methyl-succinate synthase in the case of a substituted 2methylnaphthalene (Sullivan et al. 2001). The latter is analogous to the benzoyl-CoA pathway of monoaromatic BTEX degradation. Researchers have identified several intermediates including two ring-cleaved products (Annweiler et al. 2000, 2002; Meckenstock et al. 2000, Fig. 2).

Fig. 2 Anaerobic pathways for the biodegradation of polycyclic aromatic hydrocarbons (PAHs) (adapted from Annweiler et al. 2000, 2002). A Fumarate (HOOCCH=CHCOOH), E1 naphthyl-2methyl-succinate synthase

Nitroaromatic compounds and explosives

The metabolic scheme in Fig. 3 illustrates major intermediates and end-products representative of several anaerobic TNT pathways reported to date (Esteve-Nuez et al. 2001). TNT has three highly oxidized NO2 groups at the 2,4,6-positions. Because of their electrophilic nature, these external NO2 groups are amenable to enzymatic reduction. In the meantime, since -electrons in the benzene ring are shielded by four functional groups (3NO2 and 1CH3) due to steric hindrance, the aromatic structure is very stable, preventing enzymatic attack that could lead to ring cleavage. This unique chemical structure explains, to a large extent, why biotransformation of TNT occurs rapidly but appreciable mineralization has never been achieved in either aerobic or anaerobic systems even with more than two decades of intensive research effort (Hawari et al. 2000).

Fig. 3 Anaerobic pathways for the biodegradation of nitroaromatic explosives [trinitrotoluene (TNT)] (adapted from Esteve-Nuez et al. 2001). A Bamberger rearrangement, E1 carbon monoxide dehydrogenase (CODH), E2 nitrite reductase, E3 the combination of enzymes including hydrogenase, pyruvate-ferredoxin oxidoreductase, or CODH for the first step and sulfite reductase for the final step of the reaction process (Preuss et al. 1993) An advantage of anaerobic TNT biotransformation at low redox potential is to minimize oxidative polymerization and the toxic azoxy compounds that can be readily formed in the presence of oxygen. Among an array of end-products proposed or identified (Fig. 3), the amino (NH2) and hydroxylamino (NHOH) derivatives from the reduction of NO2 groups are frequently reported. Results have also shown the removal of NO2 groups as nitrite (NO2), and the oxidation of CH3 into

benzoic acids (Esteve-Nuez and Ramos 1998; Esteve-Nuez et al. 2000). Boopathy and Kulpa (1992) even noted the formation of NH4+ from the reductive elimination of NH2 and proposed a pathway that included toluene as the transformation end-product. The role of triaminotoluene (TAT), hydroxylamino intermediates, and the resulting compounds from subsequent hydroxyl addition para to NHOH (through Bamberger rearrangement) are incompletely known under environmental conditions but have been studied in laboratory experiments (Hughes et al. 1998; 1999). TAT is considered to be a dead-end product that precludes further mineralization (Hawari et al. 2000). While hydroxylamino intermediates are not stable, their transient toxicity could be an issue in remediation systems (Tadros et al. 2000). The good news, however, is that both compounds are strongly, or even irreversibly, adsorbed to soilsa mechanism that may hold promise for remediation (Daun et al. 1998; Xue et al. 1995), and the chemically unstable nature of these compounds reduces long-term toxicity risks (Padda et al. 2000, 2003). The use of cyclodextrins for desorption of TNT-related compounds has been studied with various soils; however, the suitability of this practice over the long term is unclear (Sheremata and Hawari 2000). The enzymes involved in anaerobic TNT transformation have not been fully characterized, although several key proteins have been implicated, including ferredoxins, hydrogenases, carbon monoxide dehydrogenase (CODH), pyruvate-ferredoxin oxidoreductases, and sulfite reductase (Huang et al. 2000; Preuss et al. 1993). Perhaps more important to revitalize future research efforts is the search for new microorganisms capable of TNT ring cleavage and mineralization (Hawari et al. 2000). Unlike nitroaromatic TNT, the nonaromatic cyclic nitroamines (RDX and HMX) have weak CN bonds. Initial enzymatic attack able to change NNO2 or CH bonds can readily destabilize the cyclic structure and cause further molecular fragmentation. RDX is generally recalcitrant under aerobic conditions, therefore anaerobic metabolism has been the subject of investigation. Unfortunately, our understanding of RDX biodegradation has been limited since an early pathway study by McCormick et al. (1981). In several recent studies on the examination of approximately 24 hypothetical metabolites proposed in McCormick s pathways, only a few were confirmed, several intermediates were excluded, and many other new metabolites were identified (Adrian and Chow 2001; Hawari et al. 2000; Zhang and Hughes 2003). The full product analysis of RDX biodegradation is particularly challenging because it involves gas-phase mineralization products, unstable nitroso- and hydroxyamino intermediates, as well as small molecules such as formaldehyde and methanol. At the present time, enzymatic analysis is even more speculative despite the recent characterization of one enzyme (nitrate oxidoreductase) involved in RDX biotransformation (Bhushan et al. 2002).

Chlorinated aliphatic and aromatic hydrocarbons

The general features of anaerobic biodegradation of chlorinated compounds has been reviewed (Haggblom et al. 2000, 2003). The pathways for degradation of chlorinated aliphatic hydrocarbons (CAHs) such as PCE are well established (Fig. 4). Much remains to be understood about the biochemical mechanisms, including the enzymes and the associated genes encoding these metabolic enzymes in bacteria with various dechlorinating activities. A strain that has activity on PCE and a variety of diverse halogen compounds is Dehalococcoides ethenogenes 195 (Fennell et al. 2004; Maymo-Gatell et al. 1997). Related Dehalococcoides-like organisms have been studied

(Cupples et al. 2004; Maymo-Gatell et al 2001). Aerobic bacteria can grow on the VC intermediate of PCE degradation (Coleman et al. 2002a, b). Such information is critical so that complete PCE dechlorination can be achieved and the dechlorination rate can be maximized by maintaining optimal conditions such as redox, electron donors (normally H2), and competing electron acceptors (e.g., nitrate, sulfate).

Fig. 4 Anaerobic pathways for the biodegradation of chlorinated aliphatic tetrachloroethylene (PCE) (adapted from Cookson 1995; Rittmann and McCarty 2001). E1 PCE reductive dehydrogenase (PCE-RDase), E2 trichloroethene reductive dehydrogenase (TCE-RDase) PCE is one of the highly chlorinated (more oxidized) CAHs with no known microorganism capable of aerobic biodegradation. Due to its high electron negative character, PCE can be used as an electron acceptor (the oxidant) that is susceptible to reduction into the thermodynamically more stable VC or ethene. Reduction is accomplished either through co-metabolism (fortuitous modifications by bacteria that use other primary substrates for carbon and energy) or a novel biochemical mechanism known as dehalorespiration, where PCE is used as electron acceptor and energy generated from exergonic dehalogenation reactions is used for bacterial growth (Cookson 1995; El Fantroussi et al. 1998). The electrons needed for reductive dehalogenation of PCE are generated from the oxidation of H2 (as electron donor, Fig. 4), which originates from the fermentation of other organic compounds (DiStefano et al. 1992). Since dechlorinating bacteria compete with H2-utlilizing methanogens for H2, and a low H2 concentration is favored for dechlorinating bacteria, in practice, slow-release fermentation compounds such as fatty acids and decaying bacterial biomass are preferred (Chen 2004; Rittmann and McCarty 2001). Several enzymes and electron carriers responsible for PCE and TCE dechlorination have been characterized. Three of the four known PCE reductive dehalogenases (PCE-RDases) dechlorinate PCE or TCE to cis-DCE, but the PCE-RDase from D. ethenogenes can use PCE as sole substrate, converting it into TCE (Magnuson et al. 1998). Five chloroethene RDases have a subunit molecular mass of 5065 kDa and contain cobalamin and Fe-S clusters, and four enzymes are membrane bound (Holliger et al. 1999). TCE-RDase, located on the exterior of the cytoplasmic membrane, catalyzes the dechlorination of TCE to ethene. The gene encoding this enzyme, tceA, was cloned and sequenced via an inverse PCR approach (Magnuson et al. 2000). In studies on PCE respiration in D. multivorans, PCE dehalogenase was found in the cytoplasm and was not tightly bound to the cell membrane (Neumann et al. 1996).

The ability of anaerobic consortia (Kazumi et al. 1995) and individual organisms (Song et al. 2000, 2001) to act on chlorinated or fluorinated aromatics (Vargas et al. 2000) has been reported. Little is known about the biochemical mechanisms (particularly enzymes) of the anaerobic biodegradation of chlorinated aromatics including PCP, PCBs, and dioxins. Various anaerobic PCP pathways have been proposed, and an illustration of putative pathways is shown in Fig. 5. It is likely that bacteria may take several paths simultaneously for the removal of five chlorines leading to the formation of phenol (the rate-limiting steps) and eventually mineralization to CH4 and CO2. It is also apparent that the pathway (i.e., regiospecificity of chlorine removal) is dominated by the redox potentials and whether the bacteria are acclimated prior to PCP degradation. As can be seen from Fig. 5, certain bacteria preferentially remove chlorines in the order of para > ortho > meta (Path A, Fig. 5) (Bryant et al. 1991), whereas in others an ortho > para > meta order of chlorine removal has been reported (Path B, Fig. 5) (Mikesell and Boyd 1986). While Fig. 5 is overly simplified, a detailed description of anaerobic PCP pathways is summarized by Nicholson et al. (1992). Preferential chlorine removal has practical ramifications since some intermediates (e.g., 3,4,5-trichlorophenol) are more toxic than the parent compound, while others are possible dead-end products.

Fig. 5 Anaerobic pathways for the biodegradation of chlorinated aromatic pentachlorophenol (PCP) (adapted from Bryant et al. 1991; Mikesell and Boyd 1986). The letters o, m, p denote dechlorination at the o, m, and p positions PCBs and dioxins, although dissimilar in chemical structure, share some common features with regard to their biodegradability. PCBs contain 209 different compounds (congeners) with between 1 and 10 Cl substitutions on the backbone biphenyl structure. A typical synthetic PCB mixture contains 6080 different congeners. Dioxins have 18 Cl atoms substituted for H atoms on dibenzo-p-dioxin, giving a total of 75 possible chlorinated derivatives, the most toxic of which, i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is commonly referred to as dioxin. For both PCBs and dioxins, the less chlorinated compounds are more amenable to aerobic biodegradation. Nevertheless, reductive dechlorination is generally faster for the more highly chlorinated compounds. Anaerobic biodegradation of both PCBs and dioxins has been reported (Bunge et al.

2003) and can be enhanced by acclimation of bacteria to structurally similar, or dissimilar yet readily biodegradable, halogenated aromatic compounds, a process called priming (Deweerd and Bedard 1999; Haggblom and Young 1990, 1995; Wu et al. 1997, 1998). Early studies by Quensen et al. (1988) indicated that PCB dechlorination occurred primarily from the meta and para positions, yielding less toxic and more readily degraded products. A sequential anaerobicaerobic treatment has recently been shown to be successful in removing PCBs from contaminated soil (Master et al. 2002). The degradation pattern of PCBs is complex. Extensive meta and moderate ortho dechlorination were noted in a sediment slurry study (Wu et al. 1998), but a subsequent study using a sediment-free system indicated that bacteria specifically removed doubly flanked chlorines (i.e., chlorines bound to C that are flanked on both sides by other ClC bonds) while leaving ortho chlorines intact (Wu et al. 2000). The bacterium DF-1 dechlorinated several polychlorinated benzenes as well as PCB (Wu et al. 2002a). Like those of PCBs, the dechlorination patterns of dioxins are difficult to generalize due to the limited data available and the presence of a variety of dioxin congeners. Nevertheless, several laboratory studies and field analysis of signature compounds have all indicated predominately the initial lateral dechlorination (i.e., chlorines in the lateral 2,3,7,8 positions relative to the peri 1,4,6,9 positions), producing a characteristic 1,4-pattern of dioxin derivatives (Gaus et al. 2002; Vargas et al. 2001). This generalization, however, contrasts with recent work by Bunge et al. (2003) who proposed an initial peri-dechlorination pathway, demonstrating the diversity of dechlorinating bacteria. Although no ring cleavage has been reported thus far, dechlorination is of importance because of the reduction and even the elimination of toxicity. While the current focus is on dechlorination of highly chlorinated aromatic compounds, including PCP, PCBs and dioxin, there is less awareness within the research community of the fate and effects of the less chlorinated degradation products with higher aqueous solubility and a lower octanol/water partitioning coefficient (Mogensen et al. 2003a). In this context, dechlorination could be a blessing in disguise if it yields compounds that are more readily bioavailable and mobile (Dolfing and Beurskens 1995; Mogensen et al. 2003a).

Pesticides
The biochemical principles of pesticide biodegradation are no different from those of organic compounds discussed earlier. Although a wealth of information is available, our current understanding remains dispersed among a variety of pesticides and detailed biochemical pathways are still unknown for many pesticides, even those in common use. Nevertheless, the types of biochemical reactions are limited to a few (Alexander 1981). Under anaerobic conditions, the enzymatic reactions common to many pesticides include dechlorination, hydrolysis, nitro reduction, and dealkylation (Williams 1977). A bacterium may be partially responsible for these metabolic activities, and in some cases the bacteria may have a metabolic shift from one pathway to another (Barik et al. 1979). To illustrate, Fig. 6 describes the anaerobic reactions of three structurally distinct pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D), parathion (o,o-diethyl-o-pnitropheno phosphorothioate), and atrazine.

Fig. 6 Anaerobic pathways for the biodegradation of three selected pesticides: a 2,4dichlorophenoxyacetic acid (2,4-D), b parathion (o,o-diethyl-o-p-nitropheno phosphorothioate), and c atrazine (adapted from Crawford et al. 1998; Mikesell and Boyd 1985; Sethunathan 1973; Wackett et al. 2002). AtzA Atrazine chlorohydrase, AtzB hydroxylatrazine hydrolase, AtzC Nisopropylammelide amidohydrolase Reductive dechlorination is common to all halogenated pesticides (Fig. 6a, c), including aliphatic (fumigants), cyclic aliphatic (lindane), aromatic (DDT; PCP, Fig. 5), phenoxyalkanotes (2,4-D), aniline-based (alachlor), and cyclodiene (aldrin) (Cookson 1995). While lightly halogenated pesticides are more biodegradable under aerobic conditions, it is commonly believed that highly halogenated pesticides often biodegrade more rapidly under anaerobic conditions. Hydrolysis of phosphate esters, catalyzed by esterase, is an important mechanism for organophosphate pesticides. For example, an esterase hydrolyzed the POC linkage in parathion subsequent to a nitro reduction, which leads to the formation of p-aminophenol (Fig. 6b). Various other esterases catalyze degradation of aliphatic and aromatic ester pesticides (e.g., carbamates; Sethunathan 1973). The degradation of the nitrogen-containing pesticide atrazine shown in Fig. 6c (partially aerobic processes) involves hydrolytic dechlorination, dealkylation, and the cleavage of CN in the cyclic ring, yielding ultimate mineralization to CO2 and NH3. Anaerobic degradation of atrazine by mixed consortium and in wetlands by a co-metabolic process has been reported (Ghosh and Philip 2004; Kao et al. 2001; Seybold et al. 2001). Although N-dealkylated intermediates were not confirmed under denitrifying conditions, evidence of a hydroxyatrazine intermediate and ring cleavage was provided by Crawford et al. (1998) with the bacterial isolate M91-3. Three enzymes have been characterized in Pseudomonas sp. ADP, including atrazine chlorohydrase (AtzA), hydroxylatrazine hydrolase (AtzB), and N-iso-pylammelide amidohydrolase (AtzC) (Wackett et al. 2002).

Surfactants

For each of the three major surfactants (anionic, nonionic and cationic), current understanding of the anaerobic biochemical pathways is based on a few limited studies. Even with recent advances in sensitive analytical instrumentation, such as high resolution GC-MS, LC-MS and tandem MS, many of the putative pathways are based on a few tentatively identified intermediates. Other added challenges include cumbersome derivatization procedures, effects of sorption, difficulty in obtaining pure surfactant homologues, and the requirement for a consortium of bacteria to completely degrade a surfactant with various moieties. In this context, any detailed discussion on anaerobic surfactant degradation pathways must be speculative. Described briefly below are likely bacterial strategies in attacking nonionic and anionic surfactant moieties based on several recent studies using anaerobic microorganisms. The nonionic linear alcohol ethyoxylates (LAE) have the common structural formula CH3(CH2)mO(CH2CH2O)nH (m=717, n=125). Initial bacterial attack proposed by Steber and Wierich (1987) included the central scission at the center ether bond linking the alkyl chain with the ethoxy (EO) chaina strategy well-known for aerobic bacteria. Wagener and Schink (1988) suggested that the initial step is a hydroxyl group exchange reaction, followed by a shortening of the EO chain by stepwise cleavage of acetaldehyde. Huber et al. (2000) recently concluded that central scission is unlikely and that the first step of microbial attack is cleavage of the terminal EO unit, releasing acetaldehyde stepwise and shortening the EO until the lipophilic moiety is reached. Another major nonionic surfactant, nonylphenol ethoxylates (NPEO), has a benzene ring with an EO chain para to the C9H19 functional group. Although rapid mineralization has been reported, a recent study by Ferguson and Brownawell (2003) concluded that aromatic ring mineralization was not a major pathway for NPEO biodegradation. Anionic linear alkylbenzene sulfonate (LAS) is a mixture of related isomers and homologues consisting of a para-sulfonated benzene molecule with an alkyl chain attached to any position except the terminal one. This structural uniqueness requires the alteration of an alkyl chain, a benzene ring, and a sulfonate linkage for complete mineralization (Mogensen et al. 2003b). Under aerobic conditions, LAS biodegradation is initiated with an -oxidation of the terminal methyl group of the alkyl chain to form a carboxylic acid. Further degradation proceeds by a stepwise shortening of the alkyl chain by -oxidation, leaving a short-chain sulfophenyl carboxylic acid. The aromatic ring hydrolyzes to form a dihydroxy-benzene structure that is opened before desulfonation of the formed sulfonated dicarboxylic acid (Madsen et al. 2001). Such needed information is lacking for LAS biodegradation under various anaerobic conditions. C123LAS was desulfonated under sulphur-limited anoxic conditions (Denger and Cook 1999), suggesting that LAS may not be entirely persistent. Current data on anaerobic biodegradability does not allow an accurate survey of anaerobic biodegradation pathways of surfactants.

Practical applications of anaerobic processes in xenobiotic biodegradation

Conventional anaerobic processes have been used for the treatment of concentrated municipal and industrial wastewaters for over a century as they enjoy energy savings from methane and lower sludge production than aerobic activated sludge processes (Jewell 1987; McCarty and Smith 1986). The rapidly growing knowledge of chemical-specific bacteria and biochemical pathways

suggests that the treatment of xenobiotics, commonly at very low concentration, is technically feasible and, in many instances, also economically viable. Evidence for xenobiotic biodegradability under various anaerobic environments has stemmed predominately from lab-scale studies using serum bottles, microcosms, columns, and small-sized bioreactors. These lab-scale studies, along with many field and large-scale demonstrations are based mostly on indigenous bacteria or enriched cultures. Biodegradation tests using pure bacterial cultures or studies aimed at isolation of pure cultures (Table 1) have been almost exclusively lab-scale, although there are a few reported uses of pure bacterial inocula in pilot and field tests (e.g., Dybas et al. 1998; El Fantroussi et al. 1999). A summary in Table 2 focuses on practical applications of anaerobic bacterial consortia in field or large-scale studies and, whenever applicable, documented sources from peer reviewed journals are selected although many studies of this type are reported frequently in conference proceedings and industrial notices. Table 2 Selected large and field scale anaerobic processes in xenobiotic degradationa. BTEX Petroleum hydrocarbons (benzene-toluene-ethylbenzene-xylene), CT carbon tetrachloride, CF chloroform, UASB upflow anaerobic sludge bioreactor, CSTR continuous stirred tank reactor, APEO, alkylphenylethoxylate, AE alcohol ethyoxylate, 3-MCP 3-monochlorophenol, HCH hexachlorocyclobenzene, MCB monochlorobenzene Compounds Type and scale Performance results Reference Alkane Microcosm 44% n-alkanes removed in 12 months Salminen et al. 2004 7.5 tons hydrocarbon removed in 120 810 m Aquifer Batterman 1983 days NO2 injection stimulated BTEX BTEX Aquifer Barker et al. 1987 removal NO2 enhanced m- and p-xylene Fuel spill site Hutchins et al. 1991 removal 25 ring PAHs degraded under SO42, Rockne and Strand 1998; PAHs Microcosm NO3-reducing conditions Rothermich et al. 2002 Stimulated by humic substances; Finneran and Lovley Microcosm; MTBE field evidence in groundwater using 2001; Kolhatkar et al. groundwater 13 C of MTBE 2002 TNT to mineralizable and Full-scale reactor Funk et al. 1995 nonaromatic products by Clostridium TNT, DNT, Providing sucrose and NH4Cl, 98% RDX, HMX 51.82 m Sludge TNT, DNT and RDX removal in Lenke et al. 1998 reactor 30 weeks PCE, TCE, CT Nitrate and acetate injection Shallow aquifer Semprini et al. 1992 transformed 9597% CT to CF Significant ethene and CH4 in a TCE- McCarty and Wilson Industrial site contaminated aquifer 1992 4.5-Acre chemical Strong correlation between PCE, Major et al. 1991 plant ethene and electron donor Aquifer Nutrients, enrichment culture Ellis et al. 2000

Compounds

Type and scale

PCBs

PCP

Dioxins Chlorinated pesticide P-Based pesticide Carbamate pesticide Dinoseb pesticide Anionic surfactant Nonionic surfactant Cationic surfactant
a

Performance results Reference injection converted TCE to ethene Pseudomonas stutzeri KC removed Pilot-scale Dybas et al. 1998 CT 11% and 23% of total Cl/biphenyl 6-l Batch bioreactor Pagano et al. 1995 was reduced after 13 weeks PCP disappeared: O2-rich > O2-poor 488-m Long stream Pignatello et al. 1985 anaerobic > sorption >97.5% removal; 95% converted to Chen and Berthouex Pilot digester 3-MCP 2001 95% Removal with C source Hendriksen et al. 1992; UASB, biofilm and provided, dechlorinated at the o- then Ribarova et al. 2002; CSTR m-, but not p- chlorines; reactor Woods et al. 1989 optimization studies Dechlorinated under methanogenic Microcosm Vargas et al. 2001 conditions HCH converted to MCB and benzene In situ anaerobic which was mineralized in an aerobic Langenhoff 2003 bioscreen treatment plant Nonpersistent, degraded under PField studies Ternan et al. 1998 limited conditions Large-scale aerobic systems using 500-l Fermentor; 35 Pseudomonas sp. and recombinant Newcombe and Crowley yard3 (=26.8 m3) Escherichia coli; no large-scale 1999; Strong et al. 2000 bioreactor anaerobic processes 2,600-l Static Undetectable by 15 days after Robert et al. 1993 reactors addition of C and acclimated culture 1425% LAS12 was transformed in a Federle and Pastwa 3.5-l Digester; field CSTR reactor. Field data support 1988; Haggensen et al. data anaerobic biodegradation in 2002; Mogensen et al. sediments, landfill and soils 2003b Ferguson and Partially (APEO) to well (AE) Batch to microcosm Brownawell 2003; Huber biodegradable et al. 2000; Strongly adsorbed, toxic, scarce Batch Madsen et al. 2001 anaerobic biodegradation data

Laboratory microcosm studies were included for certain xenobiotic compounds

Compounds particularly suited to anoxic/anaerobic processes have included highly halogenated compounds such as carbon tetrachloride (CT), PCE, PCBs and some of the organochlorine pesticides that persist under aerobic conditions. Nonhalogenated compounds such as nitroaromatic and aminoaromatic compounds, including herbicides and hazardous energetic organonitro

compounds, persist under aerobic conditions and decompose only under anoxic/anaerobic conditions (Baker and Herson 1994). Morgan and Watkinson (1989) indicated that the persistent nature of compounds such as DDT and PCBs is evidence of microbial fallibility, and therefore biological cleanup of sites contaminated with this type of compound is unlikely to be generally feasible unless an extremely long treatment period is acceptable. The debate continues over whether persistent organic pollutants (POP) can be remediated by any biological means. Studies demonstrated that DDE [1,1-bis(chlorophenyl)-ethylene], a toxic byproduct of DDT, can be biodegraded into DDMU [1,1-bis(p-chlorophenyl)-2-chloro-ethylene] under methanogenic and sulfidogenic conditions (Quensen et al. 1998). DDMU has one less Cl atom and does not bioaccumulate as readily as its parent, and is also subject to dechlorination. This finding, however, was discounted by others who believe that the rate was insignificant in the field and the dechlorinating bacteria are often less favorable in competing with other bacteria (Renner 1998). Despite much success in lab studies, in practice timely remediation of POPs such as PCBs and DDT still relies heavily on non-biological means such as sediment dredging and natural capping. Chlorinated aliphatic hydrocarbons provide perhaps the most successful example of anaerobic biodegradation in anoxic aquifer environments. Under proper conditions, deliveries of electron donors and nutrients significantly stimulated the activities of reductive dechlorination in many field studies (Major et al. 1991; McCarty and Wilson 1992; Semprini et al. 1992). Field success, however, often entails expensive monitoring of the contaminant plume and the end-products including methane and ethene. In several cases where indigenous bacteria were unable to dechlorinate, bioaugmentation with pure dechlorinating bacteria has been shown to be successful (Dybas et al. 1998; Ellis et al. 2000). Field experience in remediating hydrocarbons using aerobic bacteria and pathways dates back to the early 1970s. For instance, Raymond (1974) received a patent on a process designed to remove hydrocarbon contaminants from groundwater by stimulating indigenous aerobic bacteria with nutrients and oxygen. Anaerobic processes, however, have received little attention and have had limited success in the field even with monoaromatic hydrocarbons (BTEX). Recently, field data have suggested anaerobic biodegradation could be a significant process in contaminated aquifers depleted of oxygen (Table 2). Field evidence regarding the exclusive role of anaerobes are sometimes equivocal since groundwater normally considered to be anoxic can sometimes contain dissolved oxygen (DO) as high as 1 mg/l (Batterman 1983; Hutchins et al. 1991; Steinbach et al. 2004). Future research and field demonstrations with hydrocarbons, both in terrestrial and marine environments, are likely to increase. For the terrestrial environment, research is motivated largely by the clean-up of gasoline spills in leaking underground storage tanks and the increased recognition of natural attenuation as part of the remedial strategy. For the marine environment, work is largely driven by oil spills, particularly of crude oil. Prince (1993) stated that there is room to extend current applications to oiled marshes and other anaerobic sediments as these are the frequent recipients of spill incidents. Thirty percent of gasoline sold in the United States contains 11% by volume MTBE and crude oils are composed of more than 75% aliphatic and aromatic hydrocarbons (Stocking et al. 2000). Anaerobic MTBE biodegradation is still considered to be a rare occurrence, therefore remedial applications for MTBE and other fuel oxygenates are almost exclusively aerobic processes (Fayolle et al. 2001; Stocking et al. 2000).

Anaerobic processes for the degradation of explosive compounds have been employed in both in situ and ex situ reactor systems (Funk et al. 1995; Lenke et al. 1998). Processes such as land farming, composting and slurry reactors have been very successful in transforming or detoxifying explosives and, in some cases, result in complete mineralization. Since mineralization of explosives is very unlikely in anaerobic processes, remediation is often achieved by two strategies, i.e., transformation into innocuous products or irreversible binding with soil components. Recently, increasing evidence has pointed toward the use of sequential anaerobic-aerobic processes to destroy nitroaromatic explosives (Esteve-Nuez et al. 2001; Hawari et al. 2000). The anaerobic biodegradation of pesticides and surfactants has witnessed limited in situ and ex situ applications relative to their extensive usage and disposal. Most pesticide biodegradation studies stem from the need to minimize dispersion outside of the agriculture environment, and remedial applications are limited to some contaminated pesticide manufacturing sites and accidental spills as shown in Table 2 (Langenhoff 2003; Newcombe and Crowley 1999; Roberts et al. 1993; Strong et al. 2000; Ternan et al. 1998). There is a paucity of data regarding the anaerobic biodegradation of surfactants, and surfactants commonly in use are considered as not persistent in the environment as implied from the extensive aerobic biodegradation database currently available. Surfactants are in fact the most abundant organic species in domestic sewage sludge, where concentrations exceeding g/kg levels are frequently observed (Mogensen et al. 2003a). Field monitoring data support evidence of anaerobic biodegradation in sediment below sewage treatment plant (STP) outfalls, domestic septic systems, landfill sites receiving sludge, and subsurface soils beneath laundromat wastewater discharge (Federle and Pastwa 1988). One area of needed research is the anaerobic biodegradation in sludge digesters of municipal STPs. Such anaerobic digesters are generally not designed for the removal of surfactants, hence improved designs and optimization of various anaerobic reactor systems has been the subject of several studies (Haggensen et al. 2002; Mogensen et al. 2003a). Further research is needed with regard to surfactants of current environmental concern, particularly LAS and NPEO (Ferguson and Brownawell 2003; Huber et al. 2000).

Conclusions and future prospects

The mounting evidence accumulated during the last two decades supports the argument that anaerobic biodegradation, once considered to be negligible, could be significant for a variety of xenobiotic compounds in anaerobic environments such as groundwater, sediment, landfill, sludge digesters and bioreactor systems. The elucidation of biochemical mechanisms using isolated bacteria strains, and laboratory feasibility studies using mainly enrichment cultures has enabled successful large- and field-scale in situ and ex situ remediation applications (Tables 1, 2). For certain highly chlorinated hydrocarbons (e.g., PCE), anaerobic processes cannot easily be substituted with current aerobic processes. For petroleum hydrocarbons, although aerobic processes are generally used, anaerobic biodegradation could become significant, and an economically viable option under certain circumstances (e.g., oxygen-depleted aquifer, oil-spilled marsh). For persistent compounds including PCBs, dioxins, and DDT, anaerobic processes are slow for remedial applications, but can represent a significant avenue if natural attenuation is an option. For many xenobiotic compounds, particularly PCBs and explosives, anaerobic processes could be complementary to aerobic processes for complete contaminant destruction.

With the increasing appreciation of anaerobic processes, along with recent advances in biochemical, molecular technology and analytical instrumentation, new strains will continue to be isolated and novel enzymes and biochemical pathways will be characterized. Further research will be needed to characterize genes encoding the enzymes that bacteria have evolved to degrade such xenobiotics. Recombinant strains, although still a debated issue in practice, have been explored in the case of aerobic microorganisms and show some success in outdoing the performance of indigenous bacteria (Shimazu et al. 2001; Wackett et al. 2002). Genetically engineered microorganisms capable of multiple pathways are likely to offer solutions to some of the most recalcitrant xenobiotic compounds, most likely at contained wastestreams associated with industrial facilities. An ignored area of research is the characterization of enrichment cultures. This is particularly important for recalcitrant compounds that require a consortium of syntrophic bacteria. Elucidating the ecology of these bacterial consortia is critical, but such information is almost nonexistent. A related approach involving the sequential use of anaerobic and aerobic bacteria (Esteve-Nuez et al. 2001; Lenke et al. 1998; Master et al. 2002) may also allow advances in treatment to be attained. Other knowledge gaps include the understanding and manipulation of bacterial strategies in utilizing compounds with various functional moieties. Not only the initial enzymatic attack but also the complete mineralization potential needs to be characterized. Not discussed in this review are the optimization of anaerobic processes and the provision of optimal electron donors and acceptors. Acknowledgements Research in authors laboratories has been supported by the Welch Foundation (C-1268) and BC-0022, DSWA, EIH and SERDP. This material is also based on work supported in part by the United States Army Research Laboratory and the United States Army Research Office (Grant DOD Army W911NF-04-1-0179)

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