Isolation of Adherent Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using PAH-Sorbing Carriers

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.     

Strong Impact on the Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Community of a PAH-Polluted Soil but Marginal Effect on PAH Degradation when Priming with Bioremediated Soil Dominated by Mycobacteria

Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation.     

Isolation of Marine Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Cycloclasticus Strains from the Gulf of Mexico and Comparison of Their PAH Degradation Ability with That of Puget Sound Cycloclasticus Strains

Phenanthrene- and naphthalene-degrading bacteria were isolated from four offshore and nearshore locations in the Gulf of Mexico by using a modified most-probable-number technique. The concentrations of these bacteria ranged from 10² to 10⁶ cells per ml of wet surficial sediment in mildly contaminated and noncontaminated sediments. A total of 23 strains of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were obtained. Based on partial 16S ribosomal DNA sequences and phenotypic characteristics, these 23 strains are members of the genus Cycloclasticus. Three representatives were chosen for a complete phylogenetic analysis, which confirmed the close relationship of these isolates to type strain Cycloclasticus pugetii PS-1, which was isolated from Puget Sound. PAH substrate utilization tests which included high-molecular-weight PAHs revealed that these isolates had similar, broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene. Degradation of pyrene and fluoranthene occurred only when the strains were incubated with phenanthrene. Two distinct partial PAH dioxygenase iron sulfur protein (ISP) gene sequences were PCR amplified from Puget Sound and Gulf of Mexico Cycloclasticus strains. Phylogenetic analyses of these sequences revealed that one ISP type is related to the bph type of ISP sequences, while the other ISP type is related to the nah type of ISP sequences. The predicted ISP amino acid sequences for the Gulf of Mexico and Puget Sound strains are identical, which supports the hypothesis that these geographically separated isolates are closely related phylogentically. Cycloclasticus species appear to be numerically important and widespread PAH-degrading bacteria in both Puget Sound and the Gulf of Mexico.     

Polycyclic Aromatic Hydrocarbons (PAHs) Pollution in Agricultural Soil in Tianjin,China

Representative soil samples (n = 60) collected from suburban agricultural land in Tianjin were analyzed to determine 16 PAHs in this study. Accelerated solvent extraction, GPC (Gel Permeation Chromatography), and SPE (Solid Phase Extraction) clean-up procedures were employed for PAH preparation prior to analysis with gas chromatography–mass spectrometry. The concentrations of the total PAHs (T-PAHs) ranged from 228.6 ng/g to 14722.1 ng/g with the mean value of 613.1 ng/g. Bap concentrations in many sites exceeded the suggested standards. Spatial variation of PAHs in soil was illustrated; the pollution status and comparison to other cities were also investigated. Severe PAH soil pollution was observed in some sites near urban areas. Higher PAH concentrations were detected at the downwind side of the urban areas, indicating the influence of human activities. Two indicative ratios (Fl/(Fl+Pyr, Baa/(Baa+Chr)) and principal component analysis were used to identify the possible sources of PAHs. These suggested that coal combustion was still the most important source of PAHs in Tianjin, which coincided well with the previous studies. These data can be further used to assess the health risk associated with soils polluted by PAHs and can help local government find proper ways to reduce PAHs’ pollution in soils.     

Degradation and Mineralization of the Polycyclic Aromatic Hydrocarbons Anthracene and Naphthalene in Intertidal Marine Sediments

The degradation of the polynuclear aromatic hydrocarbons (PAHs) anthracene and naphthalene by the microbiota of intertidal sediments was investigated in laboratory studies. No mineralization of either PAH was observed in the absence of oxygen. Both rates and total amounts of PAH mineralization were strongly controlled by oxygen content and temperature of the incubations. Inorganic nitrogen and glucose amendments had minimal effects on PAH mineralization. The rates and total amounts of PAH mineralized were directly related to compound concentration, pre-exposure time, and concentration. Maximum mineralization was observed at the higher concentrations (5 to 100 μg/g [ppm]) of both PAHs. Optimal acclimation to anthracene and naphthalene (through pre-exposures to the compounds) occurred at the highest acclimation concentration (1,000 ppm). However, acclimation to a single concentration (100 ppm) resulted in initial relative mineralization rates over a range of re-exposure concentrations (1 to 1,000 ppm) being nearly identical. Maximum mineralization of both PAHs occurred after intermediate periods (1 to 2 weeks) of pre-exposure. The fraction of the total heterotrophic population capable of utilizing anthracene or naphthalene as sole carbon source was also greatest after 2 weeks.     

Inhibition of Gap Junction Intercellular Communications in Cell Culture by Polycyclic Aromatic Hydrocarbons (PAH) in the Absence of PAH Metabolism

We have studied the effect of polycyclic aromatic hydrocarbons (PAH) on gap junction intercellular communications (GJIC) in culture of hepatoma cells Hep G2 and G27. Carcinogenic PAH inhibited GJIC in both cultures in contrast to non-carcinogenic PAH. We showed that both constitutive and inducible expressions of mRNAs of Ah receptor and cytochrome P4501A1 (the main isoform involved in PAH metabolism) were absent in hepatoma G27 cells. We concluded that the initial, non-metabolized molecules of carcinogenic PAH are responsible for changes in GJIC through interaction with an unknown factor in the cellular membrane.     

Evaluation of Soil Contamination by Polycyclic Aromatic Hydrocarbons in Gipuzkoa (Northern Spain)

Representative polycyclic aromatic hydrocarbons (PAHs) of low-medium molecular weight were determined using headspace solid-phase microextraction and gas chromatography with a flame ionization detector (HS-SPME-GC-FID) in ten surface soil samples from Gipuzkoa (Northern Spain). The sum of the PAHs ranged from 0.21 to 136.26 mg kg^?1. Pyrene and chrysene were the most abundant detected PAHs with an average concentration around 3.1 mg kg^?1. Pearson's correlation and PAH isomer ratios were applied to study the different origins of contamination. The results indicated that the PAH contamination in the studied area was a mixed pattern of pyrolytic and petrogenic inputs. Multivariate exploratory techniques, principal component analysis (PCA), and cluster analysis (CA) were also applied corroborating the PAH compounds patterns in the soils.     

Development of a Biokinetic Model to Evaluate Dermal Absorption of Polycyclic Aromatic Hydrocarbons from Soil

The high carcinogenic potency of polycyclic aromatic compounds often results in the dermal pathway indicating significant risk to human health at sites with contaminated soils, resulting in the establishment of conservative, risk-based remediation goals. The sorptive properties of soil sequester chemical contaminants, making them less available for uptake by receptors. Recent studies of desorption from soil indicate that PAHs follow a nonlinear desorption pattern that can be estimated by two phases: a rapid, followed by a slow, desorbing fraction. In this work, we adapt a fugacity-based model to evaluate the availability of polycyclic aromatic hydrocarbons (PAHs) from soil to human skin. Incorporating two-site desorption kinetics into the fugacity model renders a less available fraction of chemical in soil for absorption, decreasing predicted dermal uptake. We explore the impacts to dermal bioavailability of removing the “fast-desorbing” fraction of chemical from the soil. The model predicts uptake within a factor of two when compared with experimental data on dermal uptake. Soil moisture and soil loading rates emerge as potential limiting variables; however, the model is most sensitive to the size of the fast desorbing fraction of chemical in soil     

Assessment of Polycyclic Aromatic Hydrocarbons (PAH) and Heavy Metals in the Vicinity of an Oil Refinery in India

Petroleum products are one of the major sources of energy for industry and daily life. Growth of the petroleum industry and shipping of petroleum products has resulted in the pollution. Populations living in the vicinity of oil refinery waste sites may be at greater risk of potential exposure to polycyclic aromatic hydrocarbons (PAH) through inhalation, ingestion, and direct contact with contaminated media. PAH have often been found to coexist with environmental pollutants including heavy metals due to similar pollution sources. The levels and distribution patterns of Σ16 PAH (sum of the 16 PAH) and heavy metals (lead, copper, nickel, cobalt, and chromium) were determined in soil and sediment in the vicinity (5 km radius) of an oil refinery in India. Concentrations of Σ16 PAH in the soils and sediments were found to be 60.36 and 241.23 ppm, respectively. Higher amount of PAH in sediments as compared to soil is due to low water solubility of PAH, settled in the bottom of aquatic bodies. The levels of lead, copper, nickel, cobalt, and chromium (total) in soil were 12.52, 13.52, 18.78, 4.84, and 8.29 ppm, while the concentrations of these metals in sediments were 16.38, 47.88, 50.15, 7.07, and 13.25 ppm, respectively. Molecular diagnostics indices of PAH (Ratio of Phenanthrene/Anthracene, Fluranthene/Pyrene) calculated for soil and sediment samples indicate that the oil refinery environment is contaminated with PAH from petrogenic as well as pyrolytic origin and heavy vehicular traffic on the Agra- Delhi National highway. Sixteen PAH priority pollutants were detected in the United States in entire samples collected near oil refinery areas and concentrations of Σ16 PAH in soil was found to be 1.20 times higher than the threshold value for PAH in soil by ICRCL (Inter-Departmental Committee on the Redevelopment of Contaminated Land). This concentration could lead to disastrous consequences for the biotic and abiotic components of the ecosystem and may affect the soil quality, thus impairing plant growth and its bioaccumulation in food chain.     

微生物降解多环芳烃的研究进展

多环芳烃(PAHs)是具有严重危害的环境污染物质。介绍PAHs的降解菌,降解机理和PAHs的生物修复方面的研究进展。土壤中PAHs的生物修复被认为是解决污染的有效方法,目前,菲的生物降解途径已经比较清楚,但对结构更为复杂的多环芳烃研究较少。文章还对消除环境中多环芳烃的相关生物技术提出展望。     

Identification of Anthraquinone-Degrading Bacteria in Soil Contaminated with Polycyclic Aromatic Hydrocarbons

Quinones and other oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are toxic and/or genotoxic compounds observed to be cocontaminants at PAH-contaminated sites, but their formation and fate in contaminated environmental systems have not been well studied. Anthracene-9,10-dione (anthraquinone) has been found in most PAH-contaminated soils and sediments that have been analyzed for oxy-PAHs. However, little is known about the biodegradation of oxy-PAHs, and no bacterial isolates have been described that are capable of growing on or degrading anthraquinone. PAH-degrading Mycobacterium spp. are the only organisms that have been investigated to date for metabolism of a PAH quinone, 4,5-pyrenequinone. We utilized DNA-based stable-isotope probing (SIP) with [U-¹³C]anthraquinone to identify bacteria associated with anthraquinone degradation in PAH-contaminated soil from a former manufactured-gas plant site both before and after treatment in a laboratory-scale bioreactor. SIP with [U-¹³C]anthracene was also performed to assess whether bacteria capable of growing on anthracene are the same as those identified to grow on anthraquinone. Organisms closely related to Sphingomonas were the most predominant among the organisms associated with anthraquinone degradation in bioreactor-treated soil, while organisms in the genus Phenylobacterium comprised the majority of anthraquinone degraders in the untreated soil. Bacteria associated with anthracene degradation differed from those responsible for anthraquinone degradation. These results suggest that Sphingomonas and Phenylobacterium species are associated with anthraquinone degradation and that anthracene-degrading organisms may not possess mechanisms to grow on anthraquinone.     

藻类对多环芳香烃(PAHs)的富集和代谢

概述了藻类对PAHs的富集和代谢的研究进展。环境中多环芳香烃(PAHs)的污染能导致严重的健康问题,利用生物特别是微生物去除污染环境中的PAHs是一项新的技术。藻类对PAHs的富集与有机污染物的类型、藻类的种类及藻类的生物量有关,活细胞和死细胞对PAHs均有富集能力。还阐述了PAHs在真菌、细菌和藻类体内代谢的途径以及代谢过程中起关键作用的酶,PAHs在藻类中的代谢途径和细菌及真菌都不同,谷胱甘肽转移酶(GST)在藻类代谢PAH过程中起重要作用,但细胞色素P450酶所起的作用则不详。     

Polycyclic Aromatic Hydrocarbons in Road-Deposited Sediments and Roadside Soil in Tijuana,Mexico

Road-deposited sediment and roadside soil in the urban area of the city of Tijuana were collected and analyzed for 16 polycyclic aromatic hydrocarbons (PAHs). The ΣPAH concentration for road-deposited sediment (RDS) ranged between 460 and 2027 µg/kg with an average of 933 µg/kg. The ΣPAH concentration in roadside soil was lower than in RDS, with a range between 54 and 1863 µg/kg and a mean value of 308 µg/kg. The diagnostic ratios showed that the PAHs originated mostly from pyrogenic sources such as gasoline and diesel combustion. The results show that PAH concentration in RDS and roadside soils is low when compared with other published studies. The low PAH levels found in this study are possibly related to differences in climate, urban features, and anthropogenic activities conducted in the studied areas. The toxicity equivalent concentrations (TEQ) of PAH calculated were also low in comparison with other studies. This is probably due to the lower concentration of total PAHs and the highly toxic high-molecular-weight PAHs.     

Probabilistic Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Produced Water

The produced water extracted during oil and gas production includes formation water, injected water, small volumes of condensed water, and any chemical added during the oil/water separation process. Produced water contains both organic and inorganic constituents, and several studies have been conducted in the past to assess their risk. The toxicity and persistence of polycyclic aromatic hydrocarbons (PAHs) in produced water is of particular environmental concern, but there are very few studies on human health risk assessment from PAHs of produced water. This article summarizes the results of a conservative human health risk assessment approach for PAHs in produced water discharges to the marine environment. Due to the absence of available toxicity data for PAHs, the cancer slope factors were determined by using the relative potency factors (RPF) and Toxicity Equivalency Factors (TEF). Using the concentration distribution factors, the maximum cancer risks to humans were predicted in the range of 4.07 × 10^?7 to 2.95 × 10^?6. The 95th-percentile values show that the risks are well within the acceptable limits.     

Bioremediation of Polycyclic Aromatic Hydrocarbons in Creosote-Contaminated Soil by Peniophora incarnata KUC8836

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are present in products made from creosote, coal tar, and asphalt. When wood pile treated with creosote is placed in soil, PAHs can contaminate it. Creosote has been used for wood preservation in the past and is composed of approximately 85% PAHs and 15% phenolic compounds. PAHs cause harmful effects to humans and the environment because of their carcinogenic and mutagenic properties. White rot fungi can degrade not only lignin, but also recalcitrant organic compounds such as PAHs. Among numerous white rot fungi used in previous studies, four species were selected to degrade PAHs in a liquid medium. From this evaluation of the degradation of PAHs by the four fungal isolates, two species were ultimately selected for the highest rates of removal. Following 2 weeks of incubation with Peniophora incarnata KUC8836, the degradation rates of phenanthrene, fluoranthene, and pyrene were 86.5%, 77.4%, and 82.6%, respectively. Mycoaciella bispora KUC8201 showed the highest degradation rate for anthracene (61.8%). Hence, bioremediation of creosote-contaminated soil with an initial concentration of 229.49 mg kg^?1 PAHs was carried out using the two selected fungi because they could simultaneously degrade 13 more PAHs than the comparison species. More importantly, isolates of P. incarnata KUC8836 were discovered as powerful degraders of PAHs by producing laccase and manganese-dependent peroxidase (MnP), with 1.7- and 1.1-fold higher than the comparison species, respectively. Therefore, the white rot fungus may be proposed for the removal of PAHs and xenobiotic compounds in contaminated environments.     

Polycyclic Aromatic Hydrocarbon (PAH) Contamination in the Urban Topsoils of Shenyang,China

Polycyclic aromatic hydrocarbons (PAHs) in the surface urban soils of Shenyang in Northeastern China were investigated. The total concentration of the PAHs ranged from 0.09 to 8.35 mg kg^?1, with an average value of 1.51 ± 1.64 mg kg^?1. 3–5-ring PAHs accounted for 90% of total PAHs. The functional areas, such as the industrial regions (4.95 mg kg^?1) and main roads (1.56 mg kg^?1), as well as the administrative divisions, including the districts of Shenhe (1.49 mg kg^?1), Heping (2.08 mg kg^?1), and Tiexi (2.14 mg kg^?1), were heavily polluted by PAHs. The diagnostic ratios and principal component analysis (PCA) for PAHs indicate that the pollutants probably originated primarily from coal combustion and petroleum sources. The Nemerow composite index, used to assess environmental quality, shows that the soil samples were heavily polluted with PAHs, and although 52.8% of the soil sampling sites were safe, 47.2% of the soil sampling sites registered different grades of PAH pollution. The PAH contamination in Shenyang emphasizes the need for controlling fossil fuel combustion and traffic exhaust.     

Source Analysis and Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Paddy Soils

Polycyclic aromatic hydrocarbons (PAHs) are typically persistent organic pollutants with hypertoxicity and widespreading, attracting increasing attention in recent years. Six paddy land sites soils irrigated from different rivers in the Nansi Lake area of Shandong Province, China, were studied to analyze and assess the source and risk of PAHs.Analysis revealed total PAHs (TPAHs) concentrations in topsoil ranging from 57.49 to 2046.47 μg kg^?1, meanwhile, Weishan County (WS) was highly contaminated primarily from coal combustion based on the Nemerow composite index and geochemical indices.Canonical correspondence analysis (CCA) suggested that bacterial species were affected by pH, TPAHs, and soil organic matter (SOM), while covariables analysis confirmed that most effects of PAHs on bacterial diversity were attributed to the PAHs effect alone rather than the combined effects of PAHs and soil properties. The average daily human exposure (ADE) in children was approximately 2 times higher than that in adults, and the exposure paths values followed a decreasing order, oral intake > skin contact > inhalation. Furthermore, the WS site and another rice test field (TF) presented potential cancer risks and required further investigation. Therefore, the study has important theoretical significance for the control of PAHs pollution in this field, providing a scientific basis for health assessment.     

Polycyclic Aromatic Hydrocarbons (PAHs) in Surface Sediments of Lake Manzala,Egypt

This investigation represents the first extensive study of the spatial distribution, sources, and potential effects of polycyclic aromatic hydrocarbons (PAHs) in sediments from Lake Manzala, the largest of Egypt's Mediterranean coastal lakes. The concentrations of PAHs (Σ39 components) ranged from 246 to 9910 ng g^?1 dry wt., the highest values corresponding to urban hotspots with high anthropogenic input coming from wastewater discharges and combustion activities and decreasing offshore. The levels of PAHs were significantly lower compared to values reported in several coastal/estuarine areas (e.g., in Spain, Italy, USA, and Egypt) receiving substantial anthropogenic inputs from urban and industrial activities. Source ratios indicated that the PAHs were mainly from petrogenic sources in near-shore urban hotspots, with higher contributions of pyrolytic sources in coastal and offshore areas which are little influenced by human activities. Sediment quality guidelines (SQGs) showed that except at one station heavily impacted by sewage discharge, the total and individual PAH concentrations were below effect range low (ERL) concentrations that are not likely to adversely affect benthic biota.     

Kinetics of Polycyclic Aromatic Hydrocarbon (PAH) Degradation in Long-term Polluted Soils during Bioremediation

Bioremediation experiments with ten different soil samples from former industrial sites which were long-term polluted with polycyclic aromatic hydrocarbons (PAHs) were carried out using outdoor pot trials. The degradation of 15 PAHs according to the US EPA was investigated for 168 weeks through repeated soil sampling and determination of the total PAH concentration. On average, degradation was largest for acenaphthene (88%; 63 to 99%) and smallest for anthracene (22%; no significant degradation to 89%). For most of the PAH single substances, degradation kinetics were characterised by a first initial phase of fast degradation. In a subsequent second phase, degradation diminished and residual PAH concentrations were approached within 168 weeks, resulting in a similar PAH pattern in the ten soil samples. Degradation kinetics was calculated through the selection of the appropriate differential rate equation from a set of seven equations. Kinetics of PAH degradation was best fitted by single and two coupled first order exponential equations with median R2 of 0.71 (0.01 to 1.00). Degradation rate constants of the rapid phase (k ₁) ranged from 0.05×10⁻² week⁻¹ for benzo[k]fluoranthene to 18.3 week⁻¹ for naphthalene and for the subsequent slow degradation phase (k ₂) they ranged from 0.01×10⁻² week⁻¹ for benzo[a]anthracene to 2.3×10⁻² week⁻¹ for fluoranthene. Degradation was governed by desorption and diffusion processes of different rates, while microbial activity did not influence the kinetics. Median disappearance times (DT₅₀) ranged from 6.1 weeks for naphthalene to 522 weeks for benzo[k]fluoranthene. With the exception of the 6-ring PAHs dibenzo[ah]anthracene and indeno[1,2,3-cd]pyrene, this sequence followed the PAHs’ degree of condensation. The total initial PAH concentration and the residual concentration were correlated with R2 of 0.69, with larger initial PAH concentrations leading to larger residual concentrations and degradation rates.     

Influence of Vegetation on the In Situ Bacterial Community and Polycyclic Aromatic Hydrocarbon (PAH) Degraders in Aged PAH-Contaminated or Thermal-Desorption-Treated Soil

The polycyclic aromatic hydrocarbon (PAH) contamination, bacterial community, and PAH-degrading bacteria were monitored in aged PAH-contaminated soil (Neuves-Maisons [NM] soil; with a mean of 1,915 mg of 16 PAHs·kg⁻¹ of soil dry weight) and in the same soil previously treated by thermal desorption (TD soil; with a mean of 106 mg of 16 PAHs·kg⁻¹ of soil dry weight). This study was conducted in situ for 2 years using experimental plots of the two soils. NM soil was colonized by spontaneous vegetation (NM-SV), planted with Medicago sativa (NM-Ms), or left as bare soil (NM-BS), and the TD soil was planted with Medicago sativa (TD-Ms). The bacterial community density, structure, and diversity were estimated by real-time PCR quantification of the 16S rRNA gene copy number, temporal thermal gradient gel electrophoresis fingerprinting, and band sequencing, respectively. The density of the bacterial community increased the first year during stabilization of the system and stayed constant in the NM soil, while it continued to increase in the TD soil during the second year. The bacterial community structure diverged among all the plot types after 2 years on site. In the NM-BS plots, the bacterial community was represented mainly by Betaproteobacteria and Gammaproteobacteria. The presence of vegetation (NM-SV and NM-Ms) in the NM soil favored the development of a wider range of bacterial phyla (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Verrucomicrobia, Actinobacteria, Firmicutes, and Chloroflexi) that, for the most part, were not closely related to known bacterial representatives. Moreover, under the influence of the same plant, the bacterial community that developed in the TD-Ms was represented by different bacterial species (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Actinobacteria) than that in the NM-Ms. During the 2 years of monitoring, the PAH concentration did not evolve significantly. The abundance of gram-negative (GN) and gram-positive (GP) PAH-degrading bacteria was estimated by real-time PCR quantification of specific functional genes encoding the α subunit of PAH-ring hydroxylating dioxygenase (PAH-RHD_α). The percentage of the PAH-RHD_α GN bacterial genes relative to 16S rRNA gene density decreased with time in all the plots. The GP PAH-RHD_α bacterial gene proportion decreased in the NM-BS plots but stayed constant or increased under vegetation influence (NM-SV, NM-Ms, and TD-Ms).Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants associated with a wide range of anthropogenic activities (gas plants, wood preservation plants, waste incineration, coke production, and petrochemical industries). The intensive industrial coal mining during the 19th and 20th centuries in northern France caused contamination of large areas where the soil now needs to be remediated.Microbiological degradation is the chief process for natural elimination of PAHs from contaminated soil (9). A wide range of bacteria are able to degrade low-molecular-weight PAHs, such as naphthalene, phenanthrene, and anthracene, while the high-molecular-weight PAHs (with four or more fused aromatic rings) are more recalcitrant, and relatively few microorganisms are able to use them as a sole carbon source (8). The first hydroxylation step of the PAH ring is crucial to initiate an efficient biodegradation. This step is performed mainly by aerobic bacteria possessing a PAH-ring hydroxylating dioxygenase (PAH-RHD) system. Homologous PAH-RHD enzymes are encoded by specific genes present in both gram-positive (GP) and gram-negative (GN) bacterial species (22). Recently, we developed real-time PCR assays to quantify the functional genes encoding the catalytic α subunit of the PAH-RHD (PAH-RHD_α) enzyme. The quantifications were performed on soil DNA samples, giving important information about the PAH-degrading bacterial population present in various PAH-contaminated soils (7).High PAH degradation rates have been observed in laboratory experiments with strains or consortia isolated from PAH-contaminated soils (6, 33). However, in situ degradation is often a slower process due to environmental constraints and low availability of PAHs in aged, polluted soils (5, 60). As a consequence, highly contaminated soils polluted by persistent organic compounds are often treated by industrial processes such as thermal desorption (short heating of the soil at a temperature close to 500°C). Such treatment generates a soil with modified characteristics, in which the resilience of biological functions has not yet been studied. Bioremediation, the use of microorganisms to clean up contaminated soil, is an environmentally safe solution for PAH removal (20, 34) that can be accelerated by the positive effect of plants via the stimulation of microbial biodegradation in the rhizosphere (27, 44, 45, 48, 54) through root exudates (12, 39). A major driving force for the rhizosphere effect is the massive input of organic substrate in soil, which can increase the bioavailability of PAHs but also induce a selection of rhizospheric communities (17, 56) and increase the total activity, diversity, and number of bacteria (45, 48, 54, 58), as well as the abundance of PAH-degrading bacteria populations (31, 56). However, the total and functional bacterial community structure and activity in a PAH-polluted rhizosphere remain poorly described. Even if numerous studies report that plants can foster the degradation of PAHs (1, 4, 21, 28, 29, 41, 44, 45, 54), others have shown no (21) or even inhibitory (31, 55) effects of plants; thus, it is important to study the potential of rhizodegradation in situ, depending on the soil and plant studied.The aim of this study was to investigate the bacterial community density and structure, the fate of PAHs, and the PAH-degrading bacteria abundance over 2 years using a long-term in situ trial of natural and plant-assisted attenuations of PAHs. Experimental plots with contaminated soil from a former coking plant site were colonized by spontaneous vegetation (Neuves-Maisons [NM]-SV) or planted with alfalfa (Medicago sativa) (NM-Ms) and compared to the bare soil (NM-BS). Additional alfalfa-planted plots contained the same soil previously treated by thermal desorption (TD-Ms). Alfalfa was sown on the plots, since it has been shown to be effective in the removal of PAHs (46, 52, 54). Moreover, this is the first time that microbiological functions were also considered for the same soil treated by thermal desorption.