Degradation and Mineralization of High-Molecular-Weight Polycyclic Aromatic Hydrocarbons by Defined Fungal-Bacterial Cocultures

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (Penicillium janthinellum VUO 10,201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[a]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[a]pyrene was mineralized to CO₂ by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene mineralization (53% of added [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.     

Influence of cadmium and mercury on activities of ligninolytic enzymes and degradation of polycyclic aromatic hydrocarbons by Pleurotus ostreatus in soil

The white-rot fungus Pleurotus ostreatus was able to degrade the polycyclic aromatic hydrocarbons (PAHs) benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, and benzo[ghi]perylene in nonsterile soil both in the presence and in the absence of cadmium and mercury. During 15 weeks of incubation, recovery of individual compounds was 16 to 69% in soil without additional metal. While soil microflora contributed mostly to degradation of pyrene (82%) and benzo[a]anthracene (41%), the fungus enhanced the disappearance of less-soluble polycyclic aromatic compounds containing five or six aromatic rings. Although the heavy metals in the soil affected the activity of ligninolytic enzymes produced by the fungus (laccase and Mn-dependent peroxidase), no decrease in PAH degradation was found in soil containing Cd or Hg at 10 to 100 ppm. In the presence of cadmium at 500 ppm in soil, degradation of PAHs by soil microflora was not affected whereas the contribution of fungus was negligible, probably due to the absence of Mn-dependent peroxidase activity. In the presence of Hg at 50 to 100 ppm or Cd at 100 to 500 ppm, the extent of soil colonization by the fungus was limited.     

Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. ¹⁴C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of ¹⁴CO₂ recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of ¹⁴CO₂ recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in ¹⁴CO₂ production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.     

Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. ¹⁴C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of ¹⁴CO₂ recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of ¹⁴CO₂ recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in ¹⁴CO₂ production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.Bioremediation of polycyclic aromatic hydrocarbon (PAH)-polluted soil is severely hampered by the low rate of degradation of the higher PAH, particularly the four- and five-ring PAH (6, 32). These higher PAH have very low water solubility and are often tightly bound to soil particles. This results in very low bioavailability for bacterial degradation. The observation that white rot fungi can oxidize PAH rapidly with their extracellular ligninolytic enzyme systems has therefore raised interest in the use of these organisms for bioremediation of PAH-polluted soils (3, 9). Although PAHs are extensively oxidized by white rot fungi, the degree of mineralization to CO₂ is always limited. In various studies evaluating the degradation of the potent carcinogen benzo[a]pyrene by several white rot fungal species, from 0.17 to 19% of the radiolabeled PAH was recovered as ¹⁴CO₂ (4, 5, 26). The major products of the oxidation were both nonpolar and polar metabolites. The accumulation of such metabolites could be a reason for concern, since mammalian and fungal monooxygenases can oxidize benzo[a]pyrene to epoxides and dihydrodiols, which are very potent carcinogens (28, 29). However, peroxidase-mediated extracellular oxidation of benzo[a]pyrene in cultures of white rot fungi results initially in benzo[a]pyrenediones, which show weak mutagenic activity (29). These primary metabolites are rapidly oxidized further to unidentified metabolites by Phanerochaete laevis and Phanerochaete chrysosporium (5, 26). Furthermore, the oxidized benzo[a]pyrene metabolites have a higher aqueous solubility. Since the low bioavailability of PAH is a major rate-limiting factor in the degradation of these compounds by bacteria (27, 31), the increased bioavailability of oxidized PAH metabolites suggests that these compounds can be more easily mineralized by bacteria.The aim of this study was to investigate the degradation and mineralization of the five-ring PAH benzo[a]pyrene by the white rot fungus Bjerkandera sp. strain BOS55 and the subsequent mineralization of the metabolites by natural mixed cultures of microorganisms. During the oxidation and mineralization of benzo[a]pyrene, the decrease in the mutagenicity of the metabolites was monitored. The white rot fungal strain Bjerkandera sp. strain BOS55 was used because of its outstanding ability to rapidly oxidize PAH (8, 19) and because extensive information concerning its physiology is available (7, 18, 20, 22, 23).     

Formation of Bound Residues during Microbial Degradation of [14C]Anthracene in Soil

Carbon partitioning and residue formation during microbial degradation of polycyclic aromatic hydrocarbons (PAH) in soil and soil-compost mixtures were examined by using [¹⁴C]anthracenes labeled at different positions. In native soil 43.8% of [9-¹⁴C]anthracene was mineralized by the autochthonous microflora and 45.4% was transformed into bound residues within 176 days. Addition of compost increased the metabolism (67.2% of the anthracene was mineralized) and decreased the residue formation (20.7% of the anthracene was transformed). Thus, the higher organic carbon content after compost was added did not increase the level of residue formation. [¹⁴C]anthracene labeled at position 1,2,3,4,4a,5a was metabolized more rapidly and resulted in formation of higher levels of residues (28.5%) by the soil-compost mixture than [¹⁴C]anthracene radiolabeled at position C-9 (20.7%). Two phases of residue formation were observed in the experiments. In the first phase the original compound was sequestered in the soil, as indicated by its limited extractability. In the second phase metabolites were incorporated into humic substances after microbial degradation of the PAH (biogenic residue formation). PAH metabolites undergo oxidative coupling to phenolic compounds to form nonhydrolyzable humic substance-like macromolecules. We found indications that monomeric educts are coupled by C-C- or either bonds. Hydrolyzable ester bonds or sorption of the parent compounds plays a minor role in residue formation. Moreover, experiments performed with ¹⁴CO₂ revealed that residues may arise from CO₂ in the soil in amounts typical for anthracene biodegradation. The extent of residue formation depends on the metabolic capacity of the soil microflora and the characteristics of the soil. The position of the ¹⁴C label is another important factor which controls mineralization and residue formation from metabolized compounds.     

Polycyclic Aromatic Hydrocarbon Metabolism by White Rot Fungi and Oxidation by Coriolopsis gallica UAMH 8260 Laccase

We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of high levels of laccase and manganese peroxidase, was used as the growth medium. Ten fungi grown for 5 days in this medium in the presence of anthracene, pyrene, or phenanthrene, each at a concentration of 5 μg/ml could metabolize these PAHs. We studied the oxidation of 10 PAHs by using laccase purified from Coriolopsis gallica. The reaction mixtures contained 20 μM PAH, 15% acetonitrile in 60 mM phosphate buffer (pH 6), 1 mM 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and 5 U of laccase. Laccase exhibited 91% of its maximum activity in the absence of acetonitrile. The following seven PAHs were oxidized by laccase: benzo[a]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene, biphenylene, acenaphthene, and phenanthrene. There was no clear relationship between the ionization potential of the substrate and the first-order rate constant (k) for substrate loss in vitro in the presence of ABTS. The effects of mediating substrates were examined further by using anthracene as the substrate. Hydroxybenzotriazole (HBT) (1 mM) supported approximately one-half the anthracene oxidation rate (k = 2.4 h⁻¹) that ABTS (1 mM) supported (k = 5.2 h⁻¹), but 1 mM HBT plus 1 mM ABTS increased the oxidation rate ninefold compared with the oxidation rate in the presence of ABTS, to 45 h⁻¹. Laccase purified from Pleurotus ostreatus had an activity similar to that of C. gallica laccase with HBT alone, with ABTS alone, and with 1 mM HBT plus 1 mM ABTS. Mass spectra of products obtained from oxidation of anthracene and acenaphthene revealed that the dione derivatives of these compounds were present.     

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota

Human DNA polymerase ι (polι) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of polι to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to polι-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 × 10^–3 and 6 × 10^–4. Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polκ, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that polι might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polκ.     

Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability

A key issue in the nucleotide excision repair (NER) of bulky carcinogen–DNA adducts is the ability of the NER machinery to recognize and repair certain adducts while failing to repair others. Unrepaired adducts can survive to cause mutations that initiate the carcinogenic process. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region polycyclic aromatic hydrocarbon, can be metabolically activated to the enantiomeric benzo[c]phenanthrene diol epoxides (B[c]PhDEs), (+)-(1S,2R,3R,4S)-3,4- dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phe nanthrene and the corresponding (–)-(1R,2S,3S,4R) isomer. These react predominantly with adenine residues in DNA to produce the stereoisomeric 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts. Duplexes containing the 1R (+) or 1S (–) B[c]Ph-dA adduct in codon 61 of the human N-ras mutational hotspot sequence CA*A, with B[c]Ph modification at A*, are not repaired by the human NER system. However, the analogous stereoisomeric DNA adducts of the bay region benzo[a]pyrene diol epoxide (B[a]PDE), 10S (+)- and 10R (–)-trans-anti-B[a]P-N⁶-dA, are repaired in the same base sequence. In order to elucidate structural and thermodynamic origins of this phenomenon, we have carried out a 2 ns molecular dynamics simulation for the 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts in an 11mer duplex containing the human N-ras codon 61 sequence, and compared these results with our previous study of the B[a]P-dA adducts in the same sequence. The molecular mechanics Poisson– Boltzmann surface area (MM-PBSA) method was applied to calculate the free energies of the pair of stereoisomeric B[c]Ph-dA adducts, and a detailed structural analysis was carried out. The different repair susceptibilities of the B[a]P-dA adducts and the B[c]Ph-dA adducts can be attributed to different degrees of distortion, stemming from combined effects of differences in the quality of Watson–Crick hydrogen bonding, unwinding, stretching and helix backbone perturbations. These differences are due to the different intrinsic topologies of the rigid, planar bay region adducts versus the twisted, sterically hindered fjord region adducts.     

Degradation of PAHs by ligninolytic enzymes of <Emphasis Type="Italic">Irpex lacteus</Emphasis>

The ligninolytic fungus Irpex lacteus was shown as an efficient degrader of oligocyclic aromatic hydrocarbons (PAHs; 'polycyclic aromatic hydrocarbons') possessing 3-6 aromatic rings in complex liquid media. The strain produced mainly Mn-dependent peroxidase in media without pollutants. Activity of ligninolytic enzymes was higher in a N-limited medium. However, after contamination with PAHs (especially pyrene) the values increased and significant activity of Mn-independent peroxidase appeared in the complex medium. Other factors (such as the increase in nitrogen concentration or the presence of solvent(s) for dissolution of PAHs) had no effect. Cytochrome P-450 was detected in the microsomal fraction of biomass grown in the complex medium. The rate of PAH degradation was also affected by the presence of various combinations of PAHs. However, independently of the enzyme activities, anthracene was shown to have a positive influence on degradation of pyrene and fluoranthene.     

Bioremediation of PAHs-contaminated soil through composting: Influence of bioaugmentation and biostimulation on contaminant biodegradation

The degradation of several polycyclic aromatic hydrocarbons (PAHs) in soil through composting was investigated. The selected PAHs included: fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, and chrysene, with concentrations simulating a real creosote sample. The degradation of PAHs (initial concentration 1 g of total PAHs kg⁻¹ dry soil) was assessed applying bioaugmentation with the white-rot fungi Trametes versicolor and biostimulation using compost of the source-selected organic fraction of municipal solid waste (OFMSW) and rabbit food as organic co-substrates. The process performance during 30 days of incubation was evaluated through different analyses including: dynamic respiration index (DRI), cumulative oxygen consumption during 5 days (AT₅), enzymatic activity, and fungal biomass. These analyses demonstrated that the introduced T. versicolor did not significantly enhance the degradation of PAHs. However, biostimulation was able to improve the PAHs degradation: 89% of the total PAHs were degraded by the end of the composting period (30 days) compared to the only 29.5% that was achieved by the soil indigenous microorganisms without any co-substrate (control, not amended). Indeed, the results showed that stable compost from the OFMSW has a greater potential to enhance the degradation of PAHs compared to non-stable co-substrates such as rabbit food.     

糙皮侧耳和鞘脂菌NS7对多环芳烃污染土壤的生物强化与协同作用

[背景] 真菌和细菌被认为在多环芳烃污染土壤生物修复过程中发挥协同作用,目前在真实土壤体系中开展真菌-细菌协同降解研究较少。[目的] 研究真菌和细菌对不同种类多环芳烃降解的差异及对蒽和苯并[a]蒽的生物强化与协同作用。[方法] 选用多环芳烃降解真菌和细菌各一株,在液体纯培养体系下分析它们对不同种类多环芳烃降解的差异,在土壤体系中采用放射性同位素示踪技术研究2种微生物对蒽和苯并[a]蒽的生物强化与协同作用。[结果] 供试细菌鞘脂菌NS7能够很好地降解低环种类多环芳烃,以蒽作为唯一碳源时可以将其完全降解,在复合污染条件下对菲、蒽、荧蒽、芘等降解效果突出（>90%）,对苯并[a]芘降解效果较差（9.76%）。相比而言,供试真菌糙皮侧耳菌对苯并[a]芘具有更好的降解效果（21.18%）,对低环多环芳烃降解效果明显不如降解菌NS7。在自然土壤中,蒽和苯并[a]蒽具有明显不同的矿化效率,分别为18.61%和4.28%,在蒽污染土壤中加入鞘脂菌NS7并未显著提高蒽的矿化率（P>0.05）,相比而言,苯并[a]蒽污染土壤中加入糙皮侧耳显著提高了污染物矿化效率（2.24倍）,表明真菌和细菌在土壤环境中的定殖存活能力可能影响了生物强化效果。采用灭菌土壤排除土著微生物的竞争排斥作用,研究了真菌菌丝对生物强化降解的影响,发现在蒽污染土壤中,真菌菌丝的迁移作用显著提高了细菌鞘脂菌NS7对污染物的矿化率,从1.75%提高到5.91%;而在苯并[a]蒽灭菌污染土壤中,接种糙皮侧耳却没有发现苯并[a]蒽矿化率提高的现象,表明自然土壤中真菌强化降解苯并[a]蒽的作用可能是源于真菌菌丝促进污染物和土著降解菌的接触,而非直接来自真菌本身。[结论] 细菌能够很好地降解低环种类多环芳烃,而真菌对高环种类多环芳烃降解效果较好。真菌可能通过菌丝促进土著微生物在土壤中的迁移,增大多环芳烃和土著降解菌的接触,从而促进了多环芳烃降解。研究加深了对多环芳烃污染土壤生物强化修复的认识,对发展基于真菌-细菌协同作用的生物强化与调控技术提供理论指导。     

Enhancement of Solubilization and Biodegradation of Polyaromatic Hydrocarbons by the Bioemulsifier Alasan

Alasan, a high-molecular-weight bioemulsifier complex of an anionic polysaccharide and proteins that is produced by Acinetobacter radioresistens KA53 (S. Navon-Venezia, Z. Zosim, A. Gottlieb, R. Legmann, S. Carmeli, E. Z. Ron, and E. Rosenberg, Appl. Environ. Microbiol. 61:3240–3244, 1995), enhanced the aqueous solubility and biodegradation rates of polyaromatic hydrocarbons (PAHs). In the presence of 500 μg of alasan ml⁻¹, the apparent aqueous solubilities of phenanthrene, fluoranthene, and pyrene were increased 6.6-, 25.7-, and 19.8-fold, respectively. Physicochemical characterization of the solubilization activity suggested that alasan solubilizes PAHs by a physical interaction, most likely of a hydrophobic nature, and that this interaction is slowly reversible. Moreover, the increase in apparent aqueous solubility of PAHs does not depend on the conformation of alasan and is not affected by the formation of multimolecular aggregates of alasan above its saturation concentration. The presence of alasan more than doubled the rate of [¹⁴C]fluoranthene mineralization and significantly increased the rate of [¹⁴C]phenanthrene mineralization by Sphingomonas paucimobilis EPA505. The results suggest that alasan-enhanced solubility of hydrophobic compounds has potential applications in bioremediation.     

Polycyclic aromatic hydrocarbons degradation by Agrocybe sp. CU-43 and its fluorene transformation

Agrocybe sp. CU-43, a white-rot fungus isolated from Thailand, showed a high potential for degrading both low- and high-molecular weight polycyclic aromatic hydrocarbons. At 100 ppm fluorene was degraded by 99% within six days while at the same concentration 99 and 92% degradation of phenanthrene and anthracene, respectively, occurred in 21 days, and fluoranthene and pyrene were reduced by 80 and 75%, respectively, in 30 days. In a soil model, Agrocybe sp. CU-43 completely degraded 250 ppm fluorene at room temperature within four weeks. Laccase and manganese peroxidase activities, but not lignin peroxidase activity, were detected during the biodegradation of fluorene. Two of the metabolites from fluorene degradation by the fungus were identified via reversed-phase HPLC as 9-fluorenol and 9-fluorenone, the less toxic intermediates of fluorene. However, 9-fluorenol is not an end product for the degradation. These results suggest that fluorene degradation by Agrocybe sp. CU-43 may take place via the same pathway(s) employed by other ligninolytic and non-ligninolytic fungi. This is the first report of fluorene biodegradation by a fungus belonging to the genus Agrocybe.     

Nucleotide incorporation by human DNA polymerase gamma opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine

Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N²-deoxyguanosine (dG) and N⁶-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol γ) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE–dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE–dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE–dG adducts but not past BaP or BcPh DE–dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol γ opposite the BaP DE–dG adducts resembles that observed with the Y family pol η. Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases.     

Succession of Phenotypic, Genotypic, and Metabolic Community Characteristics during In Vitro Bioslurry Treatment of Polycyclic Aromatic Hydrocarbon-Contaminated Sediments

Dredged harbor sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) was removed from the Milwaukee Confined Disposal Facility and examined for in situ biodegradative capacity. Molecular techniques were used to determine the successional characteristics of the indigenous microbiota during a 4-month bioslurry evaluation. Ester-linked phospholipid fatty acids (PLFA), multiplex PCR of targeted genes, and radiorespirometry techniques were used to define in situ microbial phenotypic, genotypic, and metabolic responses, respectively. Soxhlet extractions revealed a loss in total PAH concentrations of 52%. Individual PAHs showed reductions as great as 75% (i.e., acenapthene and fluorene). Rates of ¹⁴C-PAH mineralization (percent/day) were greatest for phenanthrene, followed by pyrene and then chrysene. There was no mineralization capacity for benzo[a]pyrene. Ester-linked phospholipid fatty acid analysis revealed a threefold increase in total microbial biomass and a dynamic microbial community composition that showed a strong correlation with observed changes in the PAH chemistry (canonical r² of 0.999). Nucleic acid analyses showed copies of genes encoding PAH-degrading enzymes (extradiol dioxygenases, hydroxylases, and meta-cleavage enzymes) to increase by as much as 4 orders of magnitude. Shifts in gene copy numbers showed strong correlations with shifts in specific subsets of the extant microbial community. Specifically, declines in the concentrations of three-ring PAH moieties (i.e., phenanthrene) correlated with PLFA indicative of certain gram-negative bacteria (i.e., Rhodococcus spp. and/or actinomycetes) and genes encoding for naphthalene-, biphenyl-, and catechol-2,3-dioxygenase degradative enzymes. The results of this study suggest that the intrinsic biodegradative potential of an environmental site can be derived from the polyphasic characterization of the in situ microbial community.     

乳白耙齿菌F17对单一和复合多环芳烃的降解差异解析

[目的] 针对菲、蒽、荧蒽多环芳烃（PAHs）污染物,利用乳白耙齿菌F17,研究单一和复合PAHs污染物的生物降解规律。[方法] 采用气相色谱-质谱法（GC-MS）分析降解过程中PAHs的浓度,并采用准一级反应动力学模型对降解结果进行拟合。[结果] 对于单一PAHs,第15天时菲、蒽、荧蒽的降解率由高到低依次为菲（97.8%） > 蒽（89.3%） > 荧蒽（81.5%）。菲、蒽和荧蒽的降解过程具有准一级反应动力学特征,菲的生物降解速率最快,其次是蒽,荧蒽的降解速率最慢。与单一PAHs的降解相比,在复合PAHs的降解过程中,乳白耙齿菌F17的生长和锰过氧化物酶的合成均表现出不同的特征。此外,水溶性极可能是复合污染物降解的重要控制因子,三者水溶性为：菲 > 荧蒽 > 蒽。因此,在菲或荧蒽加入条件下,微生物能优先降解这些污染物,抑制了污染物蒽的降解;同时,蒽或菲的存在对荧蒽的降解也有抑制作用;然而外源加入水溶性较差的蒽和荧蒽,则对菲的生物降解无显著影响。[结论] 复合PAHs的生物降解主要表现为相互竞争的特点,通过GC-MS分析了PAHs的生物降解途径。     

Impact of Inoculation Protocols, Salinity, and pH on the Degradation of Polycyclic Aromatic Hydrocarbons (PAHs) and Survival of PAH-Degrading Bacteria Introduced into Soil

Degradation of polycyclic aromatic hydrocarbons (PAHs) and survival of bacteria in soil was investigated by applying different inoculation protocols. The soil was inoculated with Sphingomonas paucimobilis BA 2 and strain BP 9, which are able to degrade anthracene and pyrene, respectively. CFU of soil bacteria and of the introduced bacteria were monitored in native and sterilized soil at different pHs. Introduction with mineral medium inhibited PAH degradation by the autochthonous microflora and by the strains tested. After introduction with water (without increase of the pore water salinity), no inhibition of the autochthonous microflora was observed and both strains exhibited PAH degradation.     

Phenanthrene stimulates the degradation of pyrene and fluoranthene by <Emphasis Type="Italic">Burkholderia</Emphasis> sp. VUN10013

Pyrene and fluoranthene, when supplied as the sole carbon source, were not degraded by Burkholderia sp. VUN10013. However, when added in a mixture with phenanthrene, both pyrene and fluoranthene were degraded in liquid broth and soil. The amounts of pyrene and fluoranthene in liquid media (initial concentrations of 50 mg l⁻¹ each) decreased to 42.1% and 41.1%, respectively, after 21 days. The amounts of pyrene and fluoranthene in soil (initial concentrations of 75 mg kg⁻¹ dry soil each) decreased to 25.8% and 12.1%, respectively, after 60 days. None of the high molecular weight (HMW) polycylic aromatic hydrocarbons (PAHs) tested adversely affected phenanthrene degradation by this bacterial strain and the amount of phenanthrene decreased rapidly within 3 and 15 days of incubation in liquid broth and soil, respectively. Anthracene also stimulated the degradation of pyrene or fluoranthene by Burkholderia sp. VUN10013, but to a lesser extent than phenanthrene. The extent of anthracene degradation decreased in the presence of these HMW PAHs.     

Measurement of Monosaccharides and Conversion of Glucose to Acetate in Anoxic Rice Field Soil

Degradation of glucose has been implicated in acetate production in rice field soil, but the abundance of glucose, the temporal change of glucose turnover, and the relationship between glucose and acetate catabolism are not well understood. We therefore measured the pool sizes of glucose and acetate in rice field soil and investigated the turnover of [U-¹⁴C]glucose and [2-¹⁴C]acetate. Acetate accumulated up to about 2 mM during days 5 to 10 after flooding of the soil. Subsequently, methanogenesis started and the acetate concentration decreased to about 100 to 200 μM. Glucose always made up >50% of the total monosaccharides detected. Glucose concentrations decreased during the first 10 days from 90 μM initially to about 3 μM after 40 days of incubation. With the exception at day 0 when glucose consumption was slow, the glucose turnover time was in the range of minutes, while the acetate turnover time was in the range of hours. Anaerobic degradation of [U-¹⁴C]glucose released [¹⁴C]acetate and ¹⁴CO₂ as the main products, with [¹⁴C]acetate being released faster than ¹⁴CO₂. The products of [2-¹⁴C]acetate metabolism, on the other hand, were ¹⁴CO₂ during the reduction phase of soil incubation (days 0 to 15) and ¹⁴CH₄ during the methanogenic phase (after day 15). Except during the accumulation period of acetate (days 5 to 10), approximately 50 to 80% of the acetate consumed was produced from glucose catabolism. However, during the accumulation period of acetate, the rate of acetate production from glucose greatly exceeded that of acetate consumption. Under steady-state conditions, up to 67% of the CH₄ was produced from acetate, of which up to 56% was produced from glucose degradation.     

Biodegradation of Atrazine by Agrobacterium radiobacter J14a and Use of This Strain in Bioremediation of Contaminated Soil

We examined the ability of a soil bacterium, Agrobacterium radiobacter J14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites. J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 μg of [¹⁴C-U-ring]atrazine ml⁻¹ in 72 h with a concurrent increase in the population size from 7.9 × 10⁵ to 5.0 × 10⁷ cells ml⁻¹. Under these conditions cells mineralized the [ethyl-¹⁴C]atrazine and incorporated approximately 30% of the ¹⁴C into the J14a biomass. Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase. Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine. The addition of 10⁵ J14a cells g⁻¹ into soil with a low indigenous population of atrazine degraders treated with 50 and 200 μg of atrazine g⁻¹ soil resulted in two to five times higher mineralization than in the noninoculated soil. Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times. However, J14a introduction (10⁵ cells g⁻¹) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization.