Environmental Pollution 133 (2005) 71e84 www.elsevier.

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Principles of microbial PAH-degradation in soil

Anders R. Johnsena,1, Lukas Y. Wickb,2, Hauke Harmsb,2,*
a

National Environmental Research Institute, Department of Environmental Chemistry and Microbiology, Frederiksborgvej 399, PO Box 358, DK-4000 Roskilde, Denmark b Swiss Federal School of Technology Lausanne (EPFL), School of Architecture and Civil and Environmental Engineering, CH-1015 Lausanne, Switzerland Received 12 January 2004; accepted 13 April 2004

Capsule: Hydrophobicity and solid water distribution ratios inuence the interaction between PAHs and soil organic matter, thereby aecting microbial degradation.
Abstract Interest in the biodegradation mechanisms and environmental fate of polycyclic aromatic hydrocarbons (PAHs) is motivated by their ubiquitous distribution, their low bioavailability and high persistence in soil, and their potentially deleterious eect on human health. Due to high hydrophobicity and solidewater distribution ratios, PAHs tend to interact with non-aqueous phases and soil organic matter and, as a consequence, become potentially unavailable for microbial degradation since bacteria are known to degrade chemicals only when they are dissolved in water. As the aqueous solubility of PAHs decreases almost logarithmically with increasing molecular mass, high-molecular weight PAHs ranging in size from ve to seven rings are of special environmental concern. Whereas several reviews have focussed on metabolic and ecological aspects of PAH degradation, this review discusses the microbial PAH-degradation with special emphasis on both biological and physico-chemical factors inuencing the biodegradation of poorly available PAHs. 2004 Elsevier Ltd. All rights reserved.
Keywords: Polycyclic aromatic hydrocarbons; Biodegradation; Bioavailability; Hydrophobicity; Persistence

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are unique contaminants in the environment because they are generated continuously by the inadvertently incomplete combustion of organic matter, for instance in forest

1 Present address: Geological Survey of Denmark and Greenland, Department of Geochemistry, :ster Voldgade 10, Kbenhavn K, 1350, Denmark. 2 Present address: UFZ Centre for Environmental Research, Department of Environmental Microbiology, Permoserstrae 15, D-04318 Leipzig, Germany. Tel.: +49-341-235-2225; fax: +49-341235-2247. * Corresponding author. Tel: C41-21-6933773; fax: C41-216935670. E-mail address: hauke.harms@ufz.de (H. Harms).

res, home heating, trac, and waste incineration. Massive soil contamination with PAH originated from extensive industrial coal gasication during most of the 20th century. As gas works were typically located in densely populated urban regions to facilitate the distribution of the coal gas, PAH contaminated sites are mostly found in or near cities, thus representing a considerable public health hazard. PAHs are composed of fused, aromatic rings whose biochemical persistence arises from dense clouds of p-electrons on both sides of the ring structures, making them resistant to nucleophilic attack. Besides this, they possess physical properties, such as low aqueous solubility and high solidewater distribution ratios, which stand against their ready microbial utilization and promote their accumulation in the solid phases of the terrestrial environment. As rules of thumb, the aqueous solubility

0269-7491/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2004.04.015

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Biomass, specific growth rate, dissolution rate, dissolved concentration

and, as a consequence, the bioavailability of the PAHs decreases almost logarithmically with increasing molecular mass. Of environmental concern are primarily the PAHs ranging in size from naphthalene (two rings, C10H8) to coronene (seven rings, C24H12). This review discusses the microbial degradation of environmentally relevant PAHs with an emphasis on biological strategies to obtain poorly bioavailable, soil-sorbed or non aqueous phase PAHs.

II
Specific growth rate

III

Dissolution rate

Biomass

2. Growth on PAHs as sole carbon sources Microbial degradation of PAHs and other hydrophobic substrates is believed to be limited by the amounts dissolved in the water phase (Ogram et al., 1985; Rijnaarts et al., 1990; Volkering et al., 1992; Volkering et al., 1993; Harms and Bosma, 1997; Bosma et al., 1997), with sorbed, crystalline, and non-aqueous phase liquid (NAPL)-dissolved PAHs being unavailable to PAH-degrading organisms. Bioavailability is considered a dynamic process, determined by the rate of substrate mass-transfer to microbial cells relative to their intrinsic catabolic activity (Bosma et al., 1997; Harms and Bosma, 1997). It has been described by a bioavailability number, Bn, (Koch, 1990; Bosma et al., 1997), which is a measure of a microorganisms substrate degradation eciency in a given environment. Bn is dened as the capacity of an organisms or a populations environment to provide a chemical, divided by the capacity of the organism or population to transform that chemical. At high mass transfer rates, the overall biodegradation rate is controlled by the metabolic activity of the bacteria (Bn > 1), i.e. by both the specic activity of the cells and the population density. At Bn 1, the biodegradation rate is equally controlled by the physical transport and the microbial activity. When the transport of the substrate decreases or the bacterial population grows, the mass transfer becomes the factor that limits the biodegradation (Bn ! 1). Bacteria growing in suspended, shaken cultures with crystalline PAH in amounts exceeding the aqueous solubility as the sole source of energy and carbon exhibit characteristic growth curves (Volkering et al., 1992; Volkering et al., 1993; Wick et al., 2001a; Mulder et al., 1998), which can be divided into three phases: (i) an exponential phase, (ii) a subsequent phase with pseudolinear growth and nally (iii) a pseudo-stationary phase. A schematic illustration of the growth curves and some culture parameters is given in Fig. 1, whereas Fig. 2 shows an experimentally observed growth curve of Mycobacterium sp. LB501T with solid anthracene. The initial, exponential phase (I in Figs. 1 and 2) is similar to the exponential phase in growth curves obtained with highly water-soluble substrates. Truly exponential growth, i.e. a constant specic growth rate over several generations,

Dissolved concentration 0

Time
Fig. 1. Schematic representation of the four parameters biomass, specic growth rate, PAH dissolution rate and PAH dissolved concentration of a bacterial batch culture growing on solid PAH. A short exponential phase (I) is followed by a prolonged pseudo-linear phase (II) which approaches a pseudo-stationary phase (III). Details are given in the text.

can be explained in two ways: in one scenario, the dissolved PAH concentration saturates the bacterial uptake system. The bacterial PAH-uptake remains saturated because PAH-dissolution is fast enough to keep up with the rising substrate consumption by the growing population. In this case, bacteria grow exponentially at their physiologically limited maximum rate. When the PAH consumption by the increasing population exceeds the PAH dissolution rate, the dissolved PAH concentration drops below saturation and exponential growth ceases.

100
exponential growth pseudo-linear growth pseudo-stationary phase

I Bioavailability number (-)

II

III

2.5 2 1.5

optical density at 578 nm

10

microbial uptake limitation

1
mass transfer limitation

1 0.5

0.1

50

100

150

0 200

time (h)
Fig. 2. Batch growth (solid line) of Mycobacterium sp. LB501T on 30 g L1 of anthracene crystals of 0.2e0.5 mm as a function of theoretically calculated anthracene bioavailability expressed by the bioavailability number (Bn; dashed line) (adapted from Wick et al., 2001a). Exponential growth (I) is reected by Bn Z 1 (control by microbial activity), whereas pseudo-linear growth (II) and pseudo stationary growth (III) are reected by Bn ! 1 (mass transfer limitation).

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Alternatively, the dissolved substrate concentration may not saturate the bacterial uptake systems, but remains constant despite a growing population. In this case, exponential growth occurs at a sub-maximum rate. This is known from bacterial growth on liquid hydrocarbons and may be explained by the physical contact of the active bacteria with their substrate. The consumption-driven, local substrate dissolution remains constant per cell as long as the surface of the substrate is not lled with attached bacteria (Bouchez-Na tali et al., 2001). Whatever the mechanism, the exponential phase in the PAH growth curve is short because at some point, the potential metabolic demand of the increasing number of cells exceeds the PAH-dissolution rate (i.e., Bn drops !1). The subsequent pseudo-linear growth phase (II in Figs. 1 and 2) is explained by a physically limited, maximum dissolution of substrate which is converted into biomass. Typically, the PAH-crystals have a low area-to-volume ratio which remains constant over longer periods leading to an almost constant ux of substrate to the bacterial population. The specic growth rate decreases as the cell number increases because a growing population shares a constant total substrate ux. True linearity is unlikely because the maximum dissolution rate depends on the specic surface of the crystals, which is likely to decrease during dissolution. But the biomass formation will drop with a growing population, even when crystalline substrate is provided in excess and the dissolution rate remains constant. This is because each cell needs substrate for cell maintenance, not resulting in additional biomass. The total maintenance substrate consumption will rise with a growing population and the fraction of substrate that is channeled into biomass formation will constantly drop and approach zero (Pirt, 1965). In the resulting pseudo-stationary phase (III in Figs. 1 and 2), the substrate consumption of individual cells has reached the maintenance requirements of the cells and growth ceases. The cells are neither deprived of substrate nor inhibited by metabolic products as in the stationary phase of a classical growth curve, but they reach a quasiequilibrium where the entire PAH-ux is consumed for maintenance of the cells.

3. Growth on PAH in soil The growth curves discussed above are obtained under idealized conditions that are characterized by unlimited bacterial access to the crystals and fast substrate transport by convection and diusion in a homogeneous, aqueous solution. Heterogeneous media, such as soil, do not have these characteristics. In soil, PAHs are heterogeneously distributed and may be absorbed inside of organic particles, located in small

pores that are inaccessible for bacteria, or otherwise occluded by the multitude of solid soil constituents. In case of a massive contamination, PAHs will typically occur as tar droplets with a low surface to volume ratio, which further limits the bacterial access to the PAHs. Mixing does not occur in soil and the eective diusion of molecules in soil may be orders of magnitude lower than in water, since the diusion is retarded by the solid phases, dead-end pores and the high tortuosity of the system. Bacterial cells are excluded from pores smaller than about 0.2e0.8 mm. In addition, predation is believed to reduce bacterial biomass in pores larger than 2 mm, thereby restricting the bacterial mobility (Postma and vanVeen, 1990; Harms and Bosma, 1997). Consequently, a large fraction of the PAH-degrading bacteria in soil is expected to be physically separated from the PAH-sources, and to depend on diusive transport of PAHs from the PAH-sources to the cells (Harms and Bosma, 1997). In soil, as opposed to wellmixed aqueous systems, the substrate consumption leads much faster to mass transfer-limited conditions as the number of cells increases. Therefore, PAH-degrading populations in soil are probably mostly not growing, but they are in a pseudo-stationary phase where transient growth only replaces decaying cells until the habitats mass transfer-controlled carrying capacity is reached again. In contrast, laboratory studies with PAH-degrading, pure cultures are almost always done with PAHs in high concentrations. This is an unrealistic scenario in soil as soil-bacteria are generally believed to be carbon and energy-starved. Under soil conditions, the bacteria may not utilize a single carbon source, but rather co-utilize a number of available carbon compounds (Egli, 1995; Egli, 2002). The formation of enzymes catalyzing the degradation of organic pollutants may be repressed when the cells grow on other substrates, but the constitutive background level of expression is often sucient for immediate consumption of the pollutant if it becomes available in low amounts (Egli, 2002). As a consequence, if the cells concomitantly take up several carbon sources to maintain their biomass, there will not necessarily be a threshold concentration of PAH, below which biodegradation stops. It should be noted that the overall long-term benet to the cell of keeping a base level of PAH-degrading enzymes must be higher than the cost of producing the PAH-degrading enzymes, for the trait to be evolutionarily stable.

4. PAH-metabolism Since bacteria initiate PAH degradation by the action of intracellular dioxygenases, the PAHs must be taken up by the cells before degradation can take place. Bacteria most often oxidize PAHs to cis-dihydrodiols by

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incorporation of both atoms of an oxygen molecule. The cis-dihydrodiols are further oxidized, rst to the aromatic dihydroxy compounds (catechols) and then channeled through the ortho- or meta cleavage pathways (Cerniglia, 1984; Smith, 1990). The biological degradation of PAHs can serve three dierent functions. (i) Assimilative biodegradation that yields carbon and energy for the degrading organism and goes along with the mineralization of the compound or part of it. (ii) Intracellular detoxication processes where the purpose is to make the PAHs water-soluble as a pre-requisite for excretion of the compounds. Generally, it seems that intracellular oxidation and hydroxylation of PAHs in bacteria is an initial step preparing ring ssion and carbon assimilation, whereas in fungi it is an initial step in detoxication (Cerniglia, 1984). (iii) Co-metabolism, which is the degradation of PAHs without generation of energy and carbon for the cell metabolism. Co-metabolism is dened as a non-specic enzymatic reaction, with a substrate competing with the structurally similar primary substrate for the enzymes active site. An example is the co-metabolization of benzo(a)pyrene by bacteria growing on pyrene (Boonchan et al., 2000). Keck et al. (1989) noted that: In the case of a pure culture, co-metabolism is a dead-end transformation without benet to the organism. In a mixed culture or in the environment, however, such an initial co-metabolic transformation may pave the way for subsequent attack by another organism (Keck et al., 1989). In spite of considerable eort, only a very limited number of bacteria have been isolated that can grow in pure cultures on PAHs with ve or more aromatic rings (high molecular weight (HMW) PAHs). A possible reason is the high retention of these compounds by the solid soil phase, resulting in mass-transfer rates of HMW-PAHs to the bacterial cells too low to match the cells basic metabolic requirements. The low bioavailability of PAH may have prevented the evolution of suitable enzymatic pathways in soil bacteria. According to Perry (1979), recalcitrant compounds generally do not serve as growth substrates for any single microbial organism, but are thought to be oxidized in a series of steps by consortia of microbes. Environmental, bacterial isolates often degrade only a narrow range of PAHs (Bouchez et al., 1995) and patterns of simultaneous degradation of PAH mixtures are complex. Cometabolism and inhibition phenomena have been investigated by Bouchez et al. (1995). With mixtures of two individually degradable PAHs, either preferential degradation of one PAH or reduced degradation rates of both PAHs indicating metabolic competition were observed. The interactions were more complex when the strains were growing on one PAH (PAH1) and a second non-mineralizable PAH (PAH2) was present. (a) Degradation of PAH1 could be

unaected with no cometabolism of PAH2, (b) degradation of PAH1 could be inhibited with no cometabolism of PAH2, (c) PAH2 or derived metabolites could be toxic leading to no metabolism of PAH1, (d) degradation of PAH1 could be unaected with cometabolism of PAH2, and last, (e) cometabolism of PAH2 could have a synergistic eect increasing the degradation of PAH1. When a second bacterial strain able to degrade PAH2 was introduced, the inhibitory eects of PAH2 on strain one were relieved. The authors concluded that degradation of a PAH-mixture appears as a co-operative process involving a consortium of strains with complementary capacities. A later study (Bouchez et al., 1999) showed that mixed cultures of two or three strains, although possessing the capacity to mineralize each of ve PAHs, achieved limited degradation of a ve-PAH mixture. In contrast, an enrichment from a PAH-contaminated soil readily mineralized the ve-PAH mixture. This demonstrates that the appealing idea of releasing bacterial consortia from the lab to degrade PAH-contaminations may not be that ecient in vivo.

5. Bacterial adaptations that maximize the acquisition of sorbed and separate phase PAHs In the following, the naturally occurring, rate limiting mass transfer processes will be analysed in terms of the possibilities of biological systems to optimize the PAH transfer. 5.1. Diusion The basic assumption is that microbial degradation of PAHs is limited by the PAHs dissolved in the water phase (Volkering et al., 1992; Volkering et al., 1993; Harms and Bosma, 1997; Bosma et al., 1997). This assumption shall be considered in the context of Ficks First Law of Diusion. Diusive mass transfer strives to equalize concentration gradients. Q=t DAC0 Cx =x 1

Here, Q is the quantity of substrate (mol) diusing through area A (m2) per unit of time t (s). (C0Cx)/x is the concentration gradient where C0 is the concentration at the source (mol m3), Cx is the concentration at the sink (mol m3) and x is the diusion path length (m), i.e. the distance between source and sink. In the case of soilsorbed PAH, C0 is typically the aqueous equilibrium concentration of the PAH. D is the diusion coecient (m2 s1) expressing the resistance of the environment to diusion. The negative sign indicates that diusion is in the direction away from higher concentration. Steady state is to be expected when sorbents or NAPL provide PAHs and bacteria act as a sink, i.e., the bacterial

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Diffusion boundary

C0 x

Cx

Soil particles Sorbed or entrapped PAH

Fig. 3. Schematic drawing of a PAH-degrading bacterium growing close to soil entrapped of sorbed PAH. C0 is the PAH-concentration at the surface of the particle, Cx is the concentration at the cells and x is the diusion path length.

consumption of PAH drives the dissolution (or volatilisation through a gaseous phase) of PAH from the source (Fig. 3). From Ficks Law results that the aqueous solubility limits the potential size of the gradient. Ficks Law of Diusion also applies to bacteria grown on PAHs in shaken batch cultures. The PAHcrystals are surrounded by a stagnant boundary layer of medium through which the mass transfer occurs by diusion. The thickness of the boundary layer depends on the size and shape of the crystals and the shaking velocity (Mulder et al., 1998). The smaller the crystals and the higher the shaking velocity, the thinner is the boundary layer and the higher the PAH-ux from the crystals to the bulk liquid. For naphthalene crystals in a stirred reactor, the thickness of the boundary layer was found to vary from 10 mm to 120 mm depending on the stirring velocity (Mulder et al., 1998). We can assume uniform mixing outside the diusive boundary layer due to turbulence. If the actively growing cells are dispersed in the bulk liquid, the value of x equals the thickness of the boundary layer. 5.2. Bacterial optimization of the diusion coecient (D) and the area (A) According to Eq. (1), the PAH-ux could be increased by increasing the diusion coecient D. However, D of individual molecules is a physical parameter which cannot easily be increased by the bacteria. A biological way of increasing the eective D of PAH molecules would be the excretion of biosurfactants as carriers. In a study of the inuence of surfactants on the bioavailability of solid dibenzofuran, Garcia et al. (2001) showed that micelles of the surfactant Brij 35 facilitated the diusive transfer of dibenzofuran through the boundary layer around dibenzofuran crystals. Calculations showed, and experimental results conrmed, that the eective diusion of

micellar dibenzofuran was slightly faster than diusion of dissolved dibenzofuran. Surfactant micelles of assumed 4 ! 109 m diameter diused 7.9 times slower than a dibenzofuran molecule, while at equilibrium they contained 14.6 dibenzofuran molecules. The product of dibenzofuran solubilization and reduced diusivity of micelles resulted in 1.85 times faster eective diusion of micellar dibenzofuran. In contrast, the bioavailability of sorbed dibenzofuran was found to be reduced after addition of Brij 35. This was explained by the rate limiting intraparticle diusion of dibenzofuran that remained uninuenced by the surfactant and the reduction of the truly water-dissolved dibenzofuran concentration by solubilization in micelles (Garcia et al., 2001). Increasing the interfacial area A of the PAH-source would increase the PAH-ux (Mulder et al., 1998). For bacteria growing on alkanes or NAPL-dissolved PAH in aqueous systems, production of bioemulsiers would produce a larger surface area of these substrates and subsequently a higher substrate ux (Reddy et al., 1982). For example, addition of small amounts of biosurfactants (rhamnolipids) to Pseudomonas cultures growing on octadecane increased the octadecane dispersion by four orders of magnitude and resulted in a higher growth-rate (Zhang and Miller, 1992). Willumsen and Karlson (1997) reported that a high percentage of PAHdegraders isolated from PAH-contaminated soil had the ability to emulsify diesel fuel when grown on crystalline PAHs, but there was no correlation between PAH degradation and emulsication. Whereas emulsication of NAPL-dissolved PAH appears to be a good strategy in aqueous systems with turbulent mixing, its eect is less obvious in soil systems, where physical mixing is much smaller. 5.3. Optimization of the concentration at the sink (Cx) The concentration gradient and thus the ux of PAH can be maximized by the cells keeping the PAHconcentration at the cell-surface (Cx) close to zero (van-Loosdrecht et al., 1990; Rijnaarts et al., 1990; Harms and Bosma, 1997; Wick et al., 2001b). The rate, at which bacteria degrade a substrate that is available at low concentration, depends on their specic anity (a0A) towards the substrate. The specic anity is dened as the ratio of the maximal rate of substrate uptake (qmax) and the half saturation constant Km (Button, 1985) and equals the slope of the rst order part of the activity-versus-concentration plot. Hence, for a given substrate, a0A determines the eciency of a bacterium to reduce the substrate concentration at its surface compared to the solubility of the substrate Ceq at equilibrium. High specic anities therefore lead to ecient pollutant depletion at low concentrations at the cell surface, steeper concentration gradients, and

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higher substrate transfer rates. This strategy has been demonstrated for a Mycobacterium sp. growing on anthracene, having a specic anity for anthracene, i.e. a rst-order degradation rate constant, that was 30 times above values reported for a toluene-degrading Pseudomonas and orders of magnitude higher than bacteria growing on readily water soluble substrates (Wick et al., 2001a; Wick et al., 2002a). 5.4. Optimization of the diusion path length (x) From Ficks Law, it is seen that the PAH-ux (Q/t) is inversely proportional to the distance (x) between the PAH-source and the cells. Therefore, biolm formation on PAH-containing sorbents or separate phase PAHs is an ecient way of increasing the PAH-ux to the cells. The small distance between the PAHs and the biolm cells strongly favors the diusive mass-transfer of PAH to the cells by steepening the aqueous concentrationgradient (van-Loosdrecht et al., 1990; Rijnaarts et al., 1990). The thickness of the diusion layer around naphthalene crystals was found to be in the range of 10 to 120 mm (Mulder et al., 1998). An attached cell situated less than 1 mm from a naphthalene crystal could therefore dramatically increase the naphthalene ux to the cell compared to a cell growing in suspension. It should be noted, however, that when coupled to microbial degradation, the actual diusion ux is not inversely proportional to the distance, as Eq. (1) would suggest. Bacteria are not perfect sinks whose uptake rate can keep up with any substrate ux and the aqueous substrate concentration at the cell surface thus rises with increased mass transfer, thereby partly diminishing the gradient. Attachment to solids containing absorbed compounds was found to initially exert the same positive eect. As degradation went on, intra-particle diusion as the rate-limiting step caused increasingly substrate-depleted outer regions of the sorbent, concomitant with increasing distances between substrate and bacteria leading to reduced substrate availability (Harms and Zehnder, 1995). A recent study suggests that bacterial attachment to PAH-crystals serving as sole carbon and energy sources, may depend on the solubility of the PAH. A gfp-labeled Pseudomonas putida was grown in ow-channels with immobilized uorene or phenanthrene crystals as the source of carbon (Rodrigues et al., 2003). Confocal laser scanning microscopy revealed that the strain showed dierent behaviors depending on the kind of PAH it consumed. When grown on phenanthrene, the cells formed biolm on the crystal surfaces. In contrast, when grown on the more soluble uorene or on both uorene and phenanthrene, the strain formed biolm on the glass surface in the neighborhood of the PAH crystals, indicating that attachment to more soluble substrates was more dicult. However, the fact that phenanthrene

was not colonized in the presence of uorene, providing additional substrate, suggests that adhesion is regulated as a function of the substrate bioavailability. Such a mechanism was also suggested by Wick et al. (2001a, 2002a), who showed that the anthracene-degrading Mycobacterium strain LB501T formed a biolm on anthracene crystals. Cells at the crystal surfaces etched craters in the crystals due to consumptiondriven PAH-dissolution on a micro-scale (Wick et al., 2002a) (Fig. 4). The biolm formation by strain LB501T seems to be a well-regulated process as no biolm was formed on anthracene in the presence of alternative, soluble carbon sources or when high amounts of solid anthracene, leading to high substrate uxes, were supplied (Wick et al., 2002a). It was further found that Mycobacterium strain LB501T exhibited specic modications of the cell envelope in response to solid anthracene, such as increased adhesion to hydrophobic surfaces and changes in its mycolic acid prole (Wick et al., 2002b). This nding is of interest as mycolic acids are believed to stimulate attachment to hydrophobic

Fig. 4. Scanning electron micrograph of an anthracene surface (A) and an anthracene surface during early stages of biolm formation of Mycobacterium sp. LB501T (B). Craters were absent from fresh, untreated anthracene surfaces and thus can be attributed to anthracene consumption by surface-associated cells. Scale bars = 10 mm.

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surfaces and hence increase the access to hydrophobic substrates (Bendinger et al., 1993; Borrego et al., 2000). In another study, four sphingomonads, ve mycobacteria and a Nocardia sp. were screened for attachment to PAH-crystals during growth on phenanthrene, uoranthene, pyrene or anthracene (Johnsen and Karlson, 2004). The potential respiration of cells attached to the crystals and of cells living in the planktonic mode (i.e. in the bulk liquid) were tested with the respiration indicator WST-1. It was found for the mycobacteria and the Nocardia that the more soluble the PAH, the higher the percentage of respiration of cells in suspension. Such a split-up in attached and planktonic sub-populations was not found for the sphingomonads. Either all cells were associated with the crystals, indicating biolm formation, or all cells were planktonic. Sphingomonads and mycobacteria growing on NAPL-dissolved PAHs also adhere strongly to the NAPL phase (Pia Willumsen, personal communication). Thus, it seems that attachment to- and biolm formation on PAH-sources is a widespread mechanism among bacteria to overcome mass-transfer limitations when growing on poorly soluble and strongly sorbed PAHs. 5.5. Release of biosurfactants A bacterial strategy, which inuences the PAH transfer in a more complex way, is the release of biosurfactants. Biosurfactants are small, detergent-like molecules with a hydrophilic head and a lipophilic tail. They form spherical or lamellar micelles when the surfactant concentration exceeds a compound-specic, critical micelle concentration (CMC). Hydrophobic compounds become solubilized in the hydrophobic cores of the micelles, which leads to a transfer of PAH from solid, liquid, or sorbed PAH-pools into the water phase. In terms of Fickian diusion, biosurfactant micelles inuence the apparent solubility (C0), the eective diusion coecient (D), as was discussed above, and both the diusion distance (x) and the interfacial area (A). These eects are due to dispersion of the PAH. Surfactant molecules may furthermore inuence the dissolution or desorption process by attaching to the PAHewater interface. Here, they form hemi-micelles which may accelerate the PAH-release (for a review, see Volkering et al., 1998) Moreover, biosurfactants seem to inuence the bacterial uptake of solubilized compounds. In many instances the uptake of biosurfactant-solubilized molecules was found to be faster than the uptake of truly dissolved (i.e. monodispersed) molecules. There are furthermore reports of species-specic and energy-dependent uptake of biosurfactant-solubilized compounds, which points at a direct interaction of biosurfactant micelles with cell membranes (Beal and Betts, 2000; Noordman and Janssen, 2002). Seeing that many biosurfactants repre-

sent constituents of cell envelopes (Neu, 1996), the possibility of a fusion between micelles and cells is indeed not far-fetched. It is well known that many bacteria growing on alkanes produce biosurfactants to increase the bioavailability of these poorly available substrates (Itoh and Suzuki, 1972; Oberbremer and Muller-Hurtig, 1989). A similar strategy was suggested for PAH-degrading bacteria (Deziel et al., 1996). For example, biosurfactant produced by a Pseudomonas aeruginosa growing on phenanthrene or naphthalene was shown to increase the apparent solubility of these PAHs, suggesting that the microorganism was promoting the availability of its growth-substrate (Deziel et al., 1996). These results are in conict with earlier results showing that supernatants of a naphthalene-degrading Pseudomonas sp. culture did not aect naphthalene dissolution rates (Volkering et al., 1993). Willumsen and Karlson (1997) screened 57 PAH-degrading isolates for production of biosurfactants, but found no correlation between biosurfactant production and PAH mineralization. In a more recent study, most of 22 PAH degraders reduced the surface tension of water by only 0e4 mN m1 (Johnsen and Karlson, 2004), indicating that biosurfactants were either not produced or in concentrations far below the CMC. None of the tested sphingomonads produced biosurfactants, while a few mycobacteria and nocardia produced some biosurfactant. However, none of the tested isolates were convincing biosurfactant producers. It seems that biosurfactant production is not very common among PAH degraders and obviously not essential for obtaining PAHs under environmental conditions. The occasional accumulation of biosurfactants in aqueous cultures might be a side eect of regulated bacterial cell surface modications. Biosurfactants may change a hydrophobic surface to be hydrophilic, and vice versa (Neu, 1996). It has been suggested that these hydrophobicity/hydrophilicity shifts are a mechanism for cells to regulate attachment to- and detachment from substrata (Neu, 1996). It may be that the release of surface active compounds is a way for hydrophobic cells of Mycobacterium and Nocardia to leave hydrophobic surfaces such as PAH-sources, for instance, when cells at the bottom of the biolm become oxygen-limited or when the PAH-source they grow on is depleted. It is furthermore known that microorganisms also produce surface-active molecules for other purposes, e.g., as antibiotics (Nielsen et al., 2000; Nielsen et al., 2002).

5.6. Production of extracellular polymeric substances (EPS) The bacterial access to PAHs in the environment may be highly variable. This diculty could possibly be

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mitigated by accumulation of PAHs in times of ample supply. Organisms capable of extracting substrate pulses would have a selective advantage. This has been shown for diclofop methyl (methyl 2-[4-(2,4-dichlorophenoxy) phenoxy]pyruvate, a two-ring chlorinated herbicide) which accumulated in biolms by sorption to microbial exopolymers (Wolfaardt et al., 1994). The accumulated dichlofop was metabolized by the biolm community during starvation (Wolfaardt, 1995). The biolm regions responsible for diclofop accumulation also bound the hydrophobic, uorescent dye Nile Red, demonstrating the presence of hydrophobic binding sites (Wolfaardt and Lawrence, 1998). In another study Spath and Wuertz, 1998 found that biolm extracellular polymeric substances (EPS) contained about 60e70% of accumulated BTX (benzene, toluene, and xylenes); whereas, the BTX content in the cells was below 20%. Sorption of lipophilic PAHs to the highly hydrated and seemingly hydrophilic EPS is counterintuitive, but may be explained by hydrophobic attractions between hydrophobic and hydrophilic moieties in water (van-Oss, 1995). Sugar monomers of polysaccharides can be hydrophobic or hydrophilic, depending on their degree of hydroxylation (Neu and Poralla, 1990) and their three-dimensional conformation (van-Oss, 1995). Sorption of PAHs to EPS has indeed been reported. Twentyfour out of 28 microbial polymers tested acted as sorbents for phenanthrene and facilitated the transport of phenanthrene in sand columns (Dohse and Lion, 1994). The emulsion-stabilizing factor Alasan, excreted by Acinetobacter radiodurans, was shown to increase the aqueous solubility of PAHs (Barkay et al., 1999). We speculate that EPS may also be involved in PAH-storage in biolms. This could, for instance, be of advantage in soil where rain or bioturbation could supply microorganisms with pulses of water-dissolved PAHs. PAHdegrading biolm-organisms could then extract watersolubilized PAHs by sorption to EPS or to other cell surface constituents. In such a manner, acquired PAHs could help to survive periods of famine. A hint pointing at this is the nding that members of the genus Sphingomonas produce sphingans, a group of exopolysaccharides with a common repeating unit (Pollock, 1993), which sorb PAHs in a way that they remain readily bioavailable (Johnsen and Karlson, 2004). Although the binding of PAH did not aect the PAH transfer rates, it may result in better competitiveness of sphingan-producing organisms. It is, however, unclear whether sorption of PAHs to EPS materials serves to increase the PAH bioavailability rather than being a non-specic side eect of EPS production for other purposes. In this respect, it is known that EPS are involved in anchoring biolms to hydrophobic surfaces. EPS was for instance found to be in direct contact with anthracene crystals in Mycobacterium sp. LB501T biolms (Wick et al., 2001a) and

with phenanthrene crystals in Pseudomonas biolms (Rodrigues et al., 2003).

6. PAH-degrading bacteria are well-adapted to oligotrophic conditions prevailing in soil It has been observed that PAH degradation in soil is dominated by bacterial strains belonging to a very limited number of taxonomic groups such as Sphingomonas, Burkholderia, Pseudomonas and Mycobacterium (Kastner et al., 1994; Mueller et al., 1997; Ho et al., 2000; Bastiaens et al., 2000; Johnsen et al., 2002). Among these taxonomic groups a high proportion of the PAH-degrading isolates belong to the sphingomonads sensu lato (Mueller et al., 1997; Ho et al., 2000; Bastiaens et al., 2000; Johnsen et al., 2002), which recently have been split into the genera Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis (Takeuchi et al., 2001). Members of these genera appear to be specialized in the degradation of aromatic chemicals (e.g. Romine et al., 1999; Wattiau, 2002). Indeed, a high percentage of environmental isolates with the capability to degrade a variety of environmentally hazardous compounds, including PAHs (Frederickson et al., 1995; Mueller et al., 1997; Ho et al., 2000), dioxin compounds (Halden et al., 1999) and chlorinated phenols (Nohynek et al., 1995; Edere et al., 1997; Copley, 2000), belong to the former genus Sphingomonas. Sphingomonads are also widespread in unpolluted environments such as water distribution systems (Koskinen et al., 2000) and marine environments. In autumn samples from the northern Baltic Sea, for example, one fourth of the bacterial cells were sphingomonads (Pinhassi et al., 1997). Estuarine and marine sphingomonads are adapted to their oligotrophic habitat by having high-anity uptake systems and an ability to simultaneously take up mixed substrates (Pinhassi and Hagstrom, 2000; Schut et al., 1993; Eguchi et al., 1996; Schut et al., 1997). Some are even considered obligate oligotrophs (Cavicchioli et al., 1999; Schut et al., 1997). It seems possible that PAH-degrading sphingomonads in soil are in fact adapted to the oligotrophic environment rather than being particular specialists for the degradation of hydrophobic aromatic compounds. In this regard, it is interesting to note that the closest relative to a numerically dominant phenanthrene degrader in a PAH-polluted soil was Sphingomonas sp. strain Bal5 from the Northern Baltic Sea (Pinhassi et al., 1997; Johnsen et al., 2002). Besides being oligotrophic, a bacterium also needs the appropriate catabolic genes to be a good PAH degrader. Genes for the degradation of PAHs are often located on plasmids. Examples are the genes of the upper and lower pathways of naphthalene of several pseudomonads which are borne on iso-functional

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NAH-plasmids (Yen and Serdar, 1988). In some cases, the enzymes encoded by NAH-plasmids have broad specicities allowing the host to grow on several twoand three ring PAHs as the sole sources of carbon and energy (Foght and Westlake, 1996). A set of naphthalene degradation genes on the Pseudomonas plasmid NAH7 is part of a defective transposon, which becomes mobile in the presence of a Tn4653 transposase (Tsuda and Iino, 1990). In a Pseudomonas sp., the chromosomal gene-clusters encoding the upper- and lower naphthalene pathways were anked by genes with transposase-like sequences, suggesting that the host had acquired the naphthalene-degrading ability by transposition (Bosch et al., 1999; Bosch et al., 2000). Also, a 184 kb conjugative, catabolic plasmid from Sphingomonas aromaticivorans has been sequenced (Romine et al., 1999). Seventy-nine out of the 186 open reading frames were predicted to encode enzymes associated with the complete catabolic pathways of biphenyl, naphthalene, m-xylene and p-cresol. Other open reading frames were predicted to encode functions associated with DNA-recombination (invertase-, integrase- and transposase-like sequences), replication and transfer of the plasmid. The presence of PAH-degradative genes on mobile, genetic elements may indicate the easy spreading of PAH-catabolic abilities among bacteria in polluted soil as a result of conjugative gene-transfer. For a tarcontaminated soil, strong evidence is provided that transfer of plasmid-encoded NAH-genes has indeed occurred between phylogenetically dierent members of the bacterial community (Herrick et al., 1997; StuartKeil et al., 1998). In conclusion, it is conceivable that an ecient PAHdegrader is characterized by its intrinsic oligotrophy, a natural tendency to form biolms on surfaces in oligotrophic environments and the ability to acquire and express degradative genes.

7. Bacterial-eukaryotic consortia It is tempting to take for real that PAH degradation in soil proceeds as in laboratory assays where a pure bacterial culture or dened mixed culture mineralizes a PAH to CO2 and water without much excretion of intermediates or interference of other substrates or other organisms. In a natural setting, however, various cometabolic side-reactions will act on the PAHs and bring about a multitude of metabolites. These metabolites generally possess higher polarity than the mother compounds and one can expect that they, at least partly enter the pool of dissolved organic carbon. PAHmetabolites have also been found covalently linked to macromolecular soil organic matter (Richnow et al., 1997) and only the turnover of the organic matter would lead to the mineralization of the PAH carbon.

Numerous ligninolytic and non-ligninolytic fungi possess the ability to oxidize PAHs (Cerniglia, 1992). Since fungal mycelia constitute a large fraction of the soil biomass, they may contribute considerably to the transformation of PAH-molecules in soil. Lignin contains a variety of aromatic structures formed in plant cell walls by oxygen-radical coupling-reactions of 4-hydroxy cinnamyl, 3-methoxy-4-hydroxy cinnamyl and 3,5-dimethoxo-4-hydroxy cinnamyl, resulting in a variety of intermonomer linkages (Hammel, 1992). Ligninolytic fungi oxidize lignin extracellularly by the action of lignin peroxidases, Mn-dependent peroxidases and laccases. These enzymes are unspecic and oxidize a wide variety of organic compounds. The lack of selectivity is reasonable, considering the random structure of lignin (Hammel, 1992). The peroxidases require the presence of peroxides (e.g. hydrogen peroxide) for activity (Bollag, 1992). Electrons are removed from lignin aromatic rings to give cationic radicals, which are stabilized by cleavage of CeC bonds in the lignin skeleton. The radicals then react with O2 or water to give hydroxy- or keto-derivatives (Bollag et al., 1998). The products of the peroxidase-catalyzed PAH-oxidations are PAH-quinones (Hammel, 1992). Laccases use molecular oxygen to oxidize phenolic compounds to very reactive, free radicals (Bollag, 1992). The presence of primary, mediating substrates extend the substrate range of laccases to non-phenolic aromatics by forming potent radicals which co-oxidize non-phenolic lignin compounds (Bourbonnais and Paice, 1990) and also PAHs (Pickard et al., 1999). PAHs oxidized by fungi range in size from naphthalene to benzo(a)pyrene (Cerniglia, 1992) and a variety of white rot fungal genera are capable of oxidizing these PAHs (Cerniglia, 1984; Field et al., 1992). For instance, laccase puried from Coriolopsis gallica was shown to oxidize the structurally dierent PAHs acenaphthene, phenanthrene, biphenylene, anthracene, 2-methylanthracene, 9-methylanthracene and benzo(a)pyrene (Pickard et al., 1999). The initial attack on HMW-PAHs in soil by fungal exoenzymes appears to be more likely than attack by bacterial intracellular enzymes. Fungal exoenzymes have the advantage that they may diuse to the highly immobile HMW-PAHs. This is in contrast to bacterial PAH-dioxygenases, which are generally cell-bound because they require NADH as a co-factor. Oxidation products of PAHs are more soluble than the parent compounds and therefore more bioavailable to the microbial community as the example of the ligninolytic white rot fungus Bjerkandera sp. strain BOS55 shows (Kotterman et al., 1998). In pure-culture, 14C-benzo(a)pyrene was mobilized to a great extent (74%) by BOS55, but only limited amounts of 14CO2 were produced by the fungus. Addition of soil, sludge or LMW-PAH enrichment cultures led to a rapid increase in 14CO2

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production, indicating that some polar 14C-benzo(a)pyrene fungal metabolites were readily biodegraded, whereas others persisted. One way to increase the extent of PAH bioremediation in soil, is to mix PAH-contaminated soil with organic matter containing mycelia of white rot fungi (Lestan and Lamar, 1996). Large quantities of mycelia are available from the commercial production of fruiting bodies of several species of white rot fungi for consumption. The strategy has been investigated by mixing Pleurotus ostreatus refuse ( producing Mn-peroxidases and laccases) with creosote-contaminated soil and sh oil in microcosms under optimal conditions in the laboratory (Eggen, 1999). These experiments showed high degradation of 3-ring PAHs (89%), 4-ring PAHs (87%) and 5-ring PAHs (48%) within seven weeks. Mesocosm experiments with P. ostreatus refuse under eld conditions, however, have shown little or no reductions (Hestbjerg et al., 2003). One should be aware of possible unwanted eects of the unspecic oxidation. Some metabolites may be dead-end products which may leak into groundwater (Kotterman et al., 1998). Other fungi attack PAHs in the course of intracellular detoxication processes. During detoxication, PAHs are oxidized to epoxides by cytochrome P-450 monooxygenase (Cyt P-450). The epoxides are then nonenzymatically rearranged to phenols, which can be conjugated (Cerniglia, 1984). Alternatively, the epoxides are converted to trans-dihydrodiols by epoxide hydrolases (Cerniglia, 1984). The best studied non-ligninolytic fungus Cunninghamella elegans oxidizes numerous PAHs to phenols by oxidation at Cyt P-450 (Cerniglia, 1992). The produced epoxides are hydrolyzed to transdihydrodiols or to phenols which are subsequently conjugated with sulphate, glucoronic acid or glucose. Also, the fungal Cyt P-450 transformation of PAHs may mobilize PAHs for further degradation by bacteria. Fungalebacterial co-cultures containing the non-ligninolytic fungus Penicillium janthinelum VUO 10,201 have been shown to degrade chrysene, benzo(a)anthracene, dibenzo(a,h)anthracene and benzo(a)pyrene (Boonchan et al., 2000). Only low amounts of the PAHs were degraded when incubated with either the fungus or the bacteria. Inoculation of soil microcosms with fungale bacterial co-cultures have resulted in improved degradation of HMW-PAHs and reduced carcinogenicity of the soils (Boonchan et al., 2000). The ubiquitous co-existence of bacteria and fungi in soil and their known catabolic cooperation suggest that physical interactions between them may be of importance for PAH degradation. Recently, the formation of bacterial biolms around hyphae of mycorrhizal fungi has been demonstrated (Nurmiaho-Lassila et al., 1997; Biancotto et al., 1996; Perotto and Bonfante, 1997). It may thus be hypothesized that fungal hyphae may act as

vectors to mobilize bacteria upon fungal growth and that the creation of voids and the provision of continuous surfaces by fungal hyphae could facilitate the displacement of bacteria in soil. Multicellular animals such as nematodes, microarthropodes (e.g. springtails and mites) and annelids (earthworms) are abundant in soil. By moving around in the soil, these animals come in close contact with PAHs and may take up PAHs through the body surfaces. More important may be their PAH-uptake through feeding, since PAHs sorb to the soil organic detritus, which the animals feed on. Detoxication reactions are widespread among animals and known, e.g. in isopods (Stroomberg et al., 1999), star sh (DenBesten et al., 1992), lobsters (Li and James, 1993) and polychaetes (Driscoll and McElroy, 1996). A well-studied example is the formation of pyrene metabolites in the hepatopancreas and gut of the common soil isopod Porcellio scaber (Stroomberg et al., 1999). Pyrene was oxidized to 1-hydroxypyrene, 1-hydroxypyrene sulfate, 1-hydroxypyrene glycoside and three unidentied 1-hydroxypyrene conjugates. This was demonstrated for animals exposed to pyrene in the lab as well as eld-exposed animals. It seems likely that PAHs ingested with the food will be taken up by the soil animals, Cyt P-450-detoxied by hydroxylation and conjugation and excreted to the surroundings where it would be available to bacteria. It may be argued that heavily PAH-polluted soils are too toxic for PAH-mobilizing animals to live in, but this is certainly not the case for the diuse pollution in city centers and along roads. It may therefore be speculated that many bacteria involved in PAH degradation in soil live on eukaryotic PAH metabolites rather than on PAHs.

8. Anaerobic PAH-degradation It has been suggested that biodegradation of PAHs, both eukaryotic and prokaryotic, require the presence of molecular oxygen to initiate the enzymatic attack on the PAH molecules (Cerniglia, 1992). This would have wide implications for PAH-contamination of anaerobic sediments, water-logged soils and aquifers since contamination would practically last forever. Fortunately, there is increasing evidence of anaerobic PAH-degradation with nitrate and sulfate as terminal electron acceptors. It has recently been demonstrated with pure cultures, that anaerobic growth on naphthalene (as the sole source of carbon and energy) is possible when coupled to dissimilative nitrate reduction (Rockne et al., 2000). Two naphthalene-utilizing species were isolated from marine sediment, a denitrifying Pseudomonas sp. and a Vibrio sp. which reduced nitrate to nitrite (Rockne et al., 2000). Anaerobic PAH-degradation is not restricted to two-ring PAHs. A denitrifying enrichment culture from a uidized bed reactor showed nearly

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complete mineralization of phenanthrene (96%) (Rockne and Strand, 2001). Only small amounts of phenanthrene-derived carbon was incorporated into biomass, as opposed to 57% of the added naphthalene (Rockne and Strand, 2001). In addition, pseudomonads isolated from both contaminated and uncontaminated soils were shown to degrade phenanthrene, anthracene and pyrene under denitrifying conditions (McNally et al., 1998). PAH-degradation has also been demonstrated with sulfate as the terminal electron acceptor. Examination of the anaerobic biodegradation potential of river sediments showed that all of 15 PAHs tested were degraded to some extent (8e79%) and that PAHdegradation was probably linked to sulfate reduction (Johnson and Ghosh, 1998). In a marine, sulfatereducing sediment, uorene, phenanthrene and uoranthene were mineralized, while pyrene and benzo(a) pyrene were not (Coates et al., 1997). Sulfate-reducing enrichment cultures from an anaerobic, coal-tar-contaminated, sulfate-rich, aquifer grew with naphthalene as the sole source of carbon and energy (Bedessem et al., 1997). Addition of molybdate to the cultures, a specic inhibitor of sulfate reduction, inhibited the mineralization of naphthalene, demonstrating the link between naphthalene mineralization and sulfate reduction (Bedessem et al., 1997). Together, these studies suggest that the potential for PAH-degradation in anaerobic environments may be greater than previously recognized.

Bioaugmentation should be more than only the addition of a metabolic function. It may inuence the bioavailability of pollutants when the application methods involve homogenization, slurrying, or intensive ushing of the system, or when the bacteria added dier from the indigenous population with respect to their specic anity for the contaminant, maintenance requirements, ability to co-utilize natural substrates, active or passive mobility, adhesion behavior, or ability to produce biosurfactants and to ingest surfactantsolubilized chemicals. The bioavailability may also be aected when genetic information responsible for degradation activity of the introduced bacteria is transferred to indigenous recipient bacteria, which deviate with respect to above characteristics.

Acknowledgements We are grateful to Ulrich Karlson and Parmely Pritchard for their critical revision of the manuscript, and to all the partners of the BIOVAB/BIOSTIMUL consortia. This work was supported by the European Commission (contracts BIO4-CT97-2015 and QLRT1999-00326) and the Danish Strategic Environmental Research Program (BIOPRO).

References 9. Implications for bioaugmentation of contaminated soil with PAH-degrading bacteria Various studies have investigated the possibility of bioaugmentation of PAH-polluted soils with PAHdegrading consortia or pure strains, and enhanced PAH-degradation in soil slurries and soil-microcosms has indeed often been observed (Grosser et al., 1991; Madsen and Kristensen, 1997; Kastner et al., 1998). However, to our knowledge, enhanced PAH-biodegradation in large scale experiments as a result of inoculation with PAH-degrading lab-strains has never been demonstrated. It may be speculated that many failures of bioaugmentation in the early years of this technique may have been due to a lack of attention to the principles of PAH degradation. This negligence certainly also includes the focus on the intrinsic biochemical capacity of the inocula without consideration of their physicochemical characteristics and their capacity to colonize soil. Costerton and Lappin-Scott (1995) state: The planktonic single species laboratory culture exerts a powerful selective pressure on a bacterial genome that eventually produces a stripped down cell lacking in protective and adhesive surface structures that simply cannot survive in natural environments where adhesion and protection is of paramount importance.
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