Aerobic 10211 Bacteria

Bacteria are able to utilize PAHs as the sole source of carbon and energy (Reineke 2001). They exist as gram-positive and gram-negative bacteria with the ability to metabolize some PAHs (Cerniglia 1993).

According to Johnsen et al. (2005), bacteria growing in suspended, shaken cultures with crystalline PAHs in concentrations exceeding the aqueous solubility as the sole source of energy and carbon exhibit characteristic growth curves. These curves can be divided into three phases: exponential phase (maximum rate limited by the physiology of the bacteria, bioavailability number >1), pseudo-linear growing (maximum rate limited by the concentration of PAHs, bioavailability pyrene pyrene c/s-4,5-dihydrodiol pyrene pyrene c/s-4,5-dihydrodiol

2'-carboxiberizaldehyde 1-hydroxy2-naphthoic acid 1-hydroxy-2- 4-[1-hydroxy(2-naphthyl)-2-

Ophthalic acid phthalic acid eis- 3,4-dihydroxyphthaiic acid protocalechuic acid

3,4-dihydrodiol

Fig. 10.2 Phenanthrene biodegradation pathway in Mycobacterium species (Redrawn from Pagnout et al. 2007)

Ophthalic acid phthalic acid eis- 3,4-dihydroxyphthaiic acid protocalechuic acid

3,4-dihydrodiol

Fig. 10.2 Phenanthrene biodegradation pathway in Mycobacterium species (Redrawn from Pagnout et al. 2007)

number <1) and pseudo-stationary. However, real heterogeneous media do not have the same characteristics as these idealized conditions have.

Most of PAH-degrading bacteria oxidize PAHs using dioxygenases. A few bacteria, such as Mycobacterium sp. are also capable of oxidizing the PAHs aromatic rings via cytochrome P450 monooxygenase enzyme to form trans-dihydrodiols rather than cis-dihydrodiols (Cao et al. 2009) (Fig. 10.1). Principal well-know PAHs utilizing bacteria are shown in Table 10.2. In the Fig. 10.2, is shown a bacterial biodegradation pathway (Pagnout et al. 2007).

Some authors found that the co-metabolism is an important feature of the degradation of PAH. In some cases, there exists an inhibition on the biodegradation when two or more PAHs are presents while in other cases, an enhancement in the biodegradation is observed. Bouchez et al. (1995) conducted a study where six bacterial strains were isolated. Each of these strains was capable of using PAHs as sole carbon and energy source, at least one of them: naphthalene, phenanthrene, anthracene, fluorene, fluoranthene and pyrene. When more than one PAH were present in the test, an inhibition of the degradation occurs. They related this to PAH specific inhibitory capacity effect and solubility. Dean-Ross et al. (2002) found that Mycobacterium flavescens biodegraded fluoranthene in the presence of pyrene, although metabolization of pyrene was slower in the presence of fluoranthene than in its absence. On the other hand, Rhodococcus sp. metabolized fluoranthene in the presence of anthracene, although the presence of fluoranthene slowed the rate of anthracene biodegradation. Walter et al. (1991) found that Rhodococcus sp. UW1 could use phenanthrene, anthracene, fluoranthene and chrysene as sole sources of carbon and energy, whereas naphthalene and fluorene were only co-metabolized.

In the field of interaction between photosynthetic organisms and bacteria, Anokhina et al. (2004) studied the consumption of phenanthrene in soil by model plant-microbial associations including natural and transconjugant plasmid-bearing rhizospheric strains of Pseudomonas fluorescens and P. aureofaciens degrading polycyclic aromatic hydrocarbons. They found that the inoculation of barley seeds with both natural and transconjugant plasmid-bearing Pseudomonas strains were able to degrade PAH protecting plants from the phytotoxic action of phenanthrene and favored its degradation in soil. Yutthammo et al. (2010) investigated PAHs biodegradation by Phyllosphere bacteria of ornamental plants (mainly consisting of bacteria, such as Acinetobacter, Pseudomonas, Pseudoxanthomonas, Myco-bacterium, and uncultured bacteria). They studied ten evergreen ornamental plants: Ixora sp. (ixora), M. paniculata (orange jasmine), W. religiosa (water jasmine), Bougainvillea sp. (bougainvillea), Jasminum sambac (L.) Ait. (jasmine), Codiaeum variegatum (Croton), Ficus sp. (ficus), Streblus asper Lour. (toothbrush tree), Pseuderanthemum graciliflorum (Nees) Ridl. (blue crossandra), and Hibiscus rosa-sinensis L. (hibiscus). The results indicated that phyllosphere bacteria on unsterilized leaves, were able to enhance the activity of leaves for phenanthrene removal. Daane et al. (2001) carried out a study that indicated that the rhizosphere of salt marsh plants contained a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremedia-tion of contaminated sediments. One year later, Daane et al. (2002) described Paenibacillus naphthalenovorans sp. nov., a naphthalene-degrading bacterium from the rhizosphere of salt marsh plants. Other synergistic studies (Borde et al. 2003) have also demonstrated a relationship in algal-bacterial microcosms for the treatment of phenanthrene.

There are some studies focusing on the effect of concentration. (Mahanty et al. 2010) observed that Mycobacterium frederiksbergense degraded anthracene, naphthalene and pyrene. Experiments were conducted according to a 23 factorial design at the low (1 mg l-1) and high (50 mg l-1) levels of the PAHs in combination, in an artificial media (Bushnell Hass (BH)). The results showed that PAH removals varied 54-81% when each PAH was at low concentrations in the mixture and 67-89% at their higher concentration combinations.

Taking into consideration bioremediation studies, Das and Mukherjee (2007) found that B.subtilis DM-04 and P. aeruginosa M and NM strains could be useful in bioremediation of sites highly contaminated with crude petroleum-oil hydrocarbons. The thermophilic nature of these bacteria could add further advantage for their use in bioremediation of petroleum contaminated soils in tropical countries. Lin et al. (2010) found that a novel Bacillus fusiformis (BFN) isolated from the sludge in petroleum-contaminated wastewater can be used to effectively

Fig. 10.3 Anthracene degradation by I. lacteus (Redrawn from Catjthaml et al. 2002)

biodegrade naphthalene. It emerged that the degradation of naphthalene rose to 99.1% within 4 days under optimum conditions (temperature 30°C, pH 7.0, inoculum concentration of 0.2% and C/N ratio of 1 at initial naphthalene concentration of 50 mg/L). According to Navarro et al. (2008), Sphingomonas sp. is able to degrade anthracene, phenanthrene, pyrene and benzo[a]pyrene, in aqueous deoxyribonucleic acid solution. In their study, aqueous DNA solution was applied for the remediation of a PAH-contaminated soil by sequential soil washing and lixiviates biodegradation.

The toxicity of PAHs metabolites during bacterial degradation has been little studied. It has been reported that the metabolites of some PAHs are potentially more bioavailable and could be more toxic than the precursors. Oxy-PAHs accumulated were more toxic and more persistent than the parent compounds, highlighting the importance of complete mineralization of PAHs without accumulation of the catabolic intermediates (Cao et al. 2009).

10.2.1.2 Fungi

A diverse group of lignolytic and non-lignolytic fungi are able to oxidize PAH (Table 10.2). Fungi do not utilize PAHs as a sole source of carbon and energy, but transform PAHs co-metabolically (Cerniglia 1993).

The microbial degradation by lignolytic fungi has been intensively studied during past few years due to their ability to degrade lignin (an amorphous and complex biopolymer with an aromatic structure similar to the aromatic molecular structure of some environmental pollutants) by producing extracellular enzymes with very low substrate specificity. This makes them suitable for degradation of aromatic compounds, such as PAHs (Valentin et al. 2006; Haritash and Kaushik 2009). Fungal degradation pathway has been reflected in Fig. 10.3 (Catjthaml et al. 2002).

Non-Lignolytic Fungi

According to Reineke (2001), a variety of non-lignolytic fungi have been found to transform polycyclic aromatic hydrocarbons to metabolites that are similar to those produced by mammalian enzymes. Only a few fungi appear to have the ability to catabolize PAHs to CO2. Non-lignolytic fungi metabolize PAHs by citochrome P450 monoxygenase and epoxide hydrolase-catalysed reactions to form trans-dihydrodiols. Other metabolites formed include phenols, quinines and conjugates (Cerniglia and Sutherland 2001).

Non-lignolytic fungi, such as Cunninghamella elegans and Penicillium janth-inellum can metabolize a variety of PAHs to polar metabolites (Potin et al. 2004). Also Cladosporium sphaerospermum was able to degrade PAHs in non-sterile soils. Cunningamella elegans degraded naphthalene, acenaphthene, anthracene, phenanthrene, benzo[a]pyrene, benzo[a]anthracene, fluoranthene and pyrene (Reineke 2001). Some of these fungi that metabolize polycyclic aromatic hydrocarbons are summarized in Table 10.2, and are included into different classes: Zygomycetes, Ascomycetes, Blastomycetes, Hyphomycetes, and Coelomycetes.

Lignolytic Fungi

Ligninolytic fungi possess an extracellular degradation system which is capable of breaking down lignin (Kirk and Farrell 1987). White-rot fungi can degrade a wide range of organopollutants and the degradative activity is because of the lignin-degradating systems of these fungi (Haritash and Kaushik 2009). Many authors believe that white-rot fungi metabolize PAHs through the extracellular ligninolytic enzymes, including lignin peroxidases, Mn-peroxidase, versatile peroxidase, and laccase. The precise role of these enzymes in PAH degradation has not yet been determined; however, it has been shown that only laccase-producing fungi can mineralize PAHs to CO2 and H2O (Pozdnyakova et al. 2010).

Covino et al. (2010b) have conducted a study to assess PAH-degradation capability of L. tigrinus strain CBS 577.79 in liquid cultures, to clarify the possible involvement of laccase and MnP in the degradation process and to identify the fungal PAH degradation products. They used two media: malt extract glucose (MEG) and N- limited medium (NKLM). They obtained that Laccase activity was predominant on MEG while Mn-peroxidase (MnP) was preferentially produced in LNKM. The identification of degradation products showed the presence of several PAH derivatives, presumably derived from the action of lignin-modifying enzymes. In 2000, Márquez-Rocha (2000) found that the white rot fungus, Pleurotus ostreatus, metabolized four soil adsorbed polycyclic aromatic hydrocarbons: pyrene, anthracene and phenanthrene and were mineralized after 21 d. However, benz[a]pyrene was also oxidized, but not mineralized. They also found that biodegradation was increased when surfactant Tween 40 was added.

A factor that is necessary to take into consideration is the salinity. Valentín et al. (2006), have been studied the application of lignolytic fungi to detoxification

Xenobiotics Bacteria Fungi

Fig. 10.4 Algal transformation products of benzo[a]pyrene (Redrawn from Juhasz and Naidu 2000)

Fig. 10.4 Algal transformation products of benzo[a]pyrene (Redrawn from Juhasz and Naidu 2000)

of marine sites contaminated with PAHs, evaluating the effects of the high salinity associated with coastal areas on fungal growth, production of enzymes and ligninolytic activity. The study was carried out with four PAHs: phenanthrene, fluoranthene, pyrene and chrysene. They found an extensive inhibitory effect on PAH degradation ranging from strong for some fungi to not appreciable for others.

In biosorption context, Chen et al. (2010) carried out an experiment to elucidate biosorption of PAHs to fungal biomass and the relative contributions of biosorption and biodegradation to the total removal of PAHs by white-rot fungi. They studied five PAHs: naphthalene, acenaphthene, fluorene, phenanthrene and pyrene. In this study, the conclusion was that biosorption of PAHs by white-rot fungi is very important process influencing the fate of PAHs in the environment. The partitioning of PAHs into fungal biomass is the primary mechanism of biosorption, and sorption capabilities (Koc, carbon-normalizated partition coefficients) are linearly related to Kow (octanol-water partition coefficients).

Other factor to be taken into consideration is the culture media. Pozdnyakova et al. (2010) have carried out some experiments to study the influence of cultivation conditions on pyrene degradation by the fungus Pleurotus ostreatus D1. They found that in Kirk's medium, about 65.6 ± 0.9% of the initial pyrene was metabolized after three weeks, and that in basidiomycetes rich medium, P. ostreatus D1 metabolized up to 89.8 ± 2.3% of pyrene within three weeks. In the first case, pyrene-4,5-dihydrodiol was accumulated where as it did not exist in the latter case. Experiments were also conducted to determine the effect of fungal inoculum, biomass and glucose concentrations on fluoranthene, pyrene and chrysene degradation by Bjerkandera sp. BOS55 (Valentín et al. 2007). This work showed the capability of the white-rot fungus to degrade PAHs in soil slurry phase

Chrysene Biotransformation

Phenol

Fig. 10.5 Proposed pathways for anaerobic biotransformation of phenanthrene by sulfate-reducing bacteria (Redrawn from Tesai et al. 2009)

Phenol

Fig. 10.5 Proposed pathways for anaerobic biotransformation of phenanthrene by sulfate-reducing bacteria (Redrawn from Tesai et al. 2009)

bioreactors. They found that the use of free mycelia seems to be beneficial for the degradative action of the fungus. Thus, degradation of the PAHs was enhanced between 13 and 29% by using mycelium as inoculum compared to fermentation with pellets inoculation. They also found that a small change in the initial glucose concentration in the range 20-3 g/L did not exert a significant effect on PAH degradation while the initial biomass exerted a larger effect on PAH degradation compared to the other two studied parameters. In the study of very concentrated media, Bumpus (1989) demonstrated that P. chrysosporium is able to degrade PAHs present in anthracene oil (heavy oil which distils over from coal tar). Analysis by capillary gas chromatography and high-performance liquid chroma-tography showed that at least 22 PAHs, including all of the most abundant PAH components present in anthracene oil, underwent 70-100% disappearance during 27 days of incubation under nutrient nitrogen-limited media. An experiment was conducted by Kotterman et al. (1994) to optimize the biodegradation of anthracene by Bjerkandera sp. with respect to O2, N, and C. They concluded that the supply of O2 was the most important factor in the biodegradation of anthracene.

Bioremediation of polluted liquid and solid matrices with lignolytic fungi has been widely studied for last several years. There are some works on comparison of different organisms. A study to assess the PAH-biodegradation potential of L. tigrinus CBS 577.79 on real solid matrices derived from a wood treatment plant in view of its possible use in mycoremediation applications was carried out by Covino et al. (2010a). They compared the results with Irpex lacteus. They found that L. tigrinus was able to colonize and detoxify solid PAH-contaminated matrices under non-sterile conditions. Its growth and degradation performances were invariably higher than those of I. lacteus.

Like in other situations, the factor of the aged in the matrix is important. Eggen and Majcherczyk (1998) have studied the degradation and metabolisms of benzo[a]pyrene in soil inoculated with P. ostreatus. Two strains of P. ostreatus were used in a mineralization study with [14C]benzo[a]pyrene. They observed that in aged soil contaminated with creosote, P. ostreatus removed benzo[a]pyrene most extensively in the very first month. Elimination of spiked 14C-benzo[a]pyrene was higher than benzo[a]pyrene originally present, and 40% removal of the first compound was observed. Although mineralization was low (1%), but there was a significant inoculation effect (with white rot fungi) on mineralization as compared to control.

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