Comparing Anthracene and Fluorene Degradation in Anthracene and Fluorene-Contaminated Soil by Single and Mixed Plant Cultivation

The ability of three plant species (sweet corn, cucumber, and winged bean) to remediate soil spiked with 138.9 and 95.9 mg of anthracene and fluorene per kg of dry soil, respectively, by single and double plant co-cultivation was investigated. After 15 and 30 days of transplantation, plant elongation, plant weight, chlorophyll content, and the content of each PAH in soil and plant tissues were determined. Based on PAH removal and plant health, winged bean was the most effective plant for phytoremediation when grown alone; percentage of fluorene and anthracene remaining in the rhizospheric soil after 30 days were 7.8% and 24.2%, respectively. The most effective combination of plants for phytoremediation was corn and winged bean; on day 30, amounts of fluorene and anthracene remaining in the winged bean rhizospheric soil were 3.4% and 14.3%, respectively; amounts of fluorene and anthracene remaining in the sweet corn rhizospheric soil were 4.1% and 8.8%, respectively. Co-cultivation of sweet corn and cucumber could remove fluorene to a higher extent than anthracene from soil within 15 days, but these plants did not survive and died before day 30. The amounts of fluorene remaining in the rhizospheric soil of corn and cucumber were only 14% and 17.3%, respectively, on day 15. No PAHs were detected in plant tissues. This suggests that phytostimulation of microbial degradation in the rhizosphere was most likely the mechanism by which the PAHs were removed from the spiked soil. The results show that co-cultivation of plants has merit in the phytoremediation of PAH-spiked soil.     

REDUCTION IN SOIL POLYCYCLIC AROMATIC HYDROCARBONS BY ARBUSCULAR MYCORRHIZAL LEEK PLANTS

The effect of arbuscular mycorrhizal fungi (AMF) on the reduction of soil polycyclic aromatic hydrocarbon (PAH), nutrient uptake, and growth of leek (Allium porrum L. cv. Musselburgh) plants was studied under greenhouse conditions. This experiment was a 3 × 2 × 2 × 4 factorial design including three mycorrhizal treatments (non-AMF, Glomus intraradices, and G. versiforme strains), two microorganism statuses (with and without soil bacteria), two PAH chemicals (anthracene and phenanthrene), and four PAH concentrations (three concentrations added and one control). Leek growth was reduced significantly in soils spiked with anthracene or phenanthrene. Inoculation with either Glomus intraradices or G. versiforme not only increased N and P uptake and plant growth, but also enhanced PAH disappearance in soil. After 12 weeks of potcultures, the anthracene and phenanthrene concentrations in soils were decreased as compared to their initial level, 9%–31% versus 43%–88%, respectively. Reductions in concentration were larger for phenanthrene than anthracene. The addition of a soil microorganism (SM) extract in potcultures accelerated the disappearance of PAHs. The decrease of PAHs in soil was mainly attributed to the enhanced nutrient uptake by AMF, leading to improved plant growth, which, in turn, may stimulate soil microbial activity. This study shows the interrelationships between AMF, plants, other SMs, and PAH disappearance in soil. The phytoremediation of soil contaminated with PAHs can be accelerated through inoculation with AMF and other SMs.     

Degradation of High Molecular Weight PAHs in Contaminated Soil by a Bacterial Consortium: Effects on Microtox and Mutagenicity Bioassays

Bioaugmentation of polycyclic aromatic hydrocarbon (PAH)-contaminated soil was investigated using a mixed bacterial culture (community five) isolated from an abandoned industrial site. Community five was inoculated into contaminated soil containing a total PAH (two- to five-ring compounds) concentration of approximately 820 mg/kg soil. PAH degradation by the indigenous microbial population was restricted to the lower molecular weight compounds (naphthalene, acenaphthene, fluorene and phenanthrene) even with yeast extract addition: these compounds decreased by 14 to 37%, in soil hydrated to 50% water capacity, following 91 days of incubation at 24°C. Inoculation of community five into this PAH-contaminated soil resulted in significant decreases in the concentration of all PAHs over the incubation period: greater than 86% of naphthalene, acenaphthene, fluorene, and phenanthrene were degraded after 91 days, while anthracene, fluoranthene, and pyrene were degraded to lesser extents (51.7 to 57.6%). A lag period of 48 to 63 days was observed before the onset of benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene removal. However, significant decreases in the concentration of these compounds (32.6, 25.2, and 18.5%, respectively) were observed after 91 days. No significant decrease in the mutagenic potential of organic soil extracts (as measured by the Ames Test) was observed after incubation of the soil with the indigenous microflora; however, the Microtox toxicity of aqueous soil extracts was reduced sevenfold. In contrast, extracts from contaminated soil inoculated with community five underwent a 43% decrease in mutagenic potential and the toxicity was reduced 170-fold after 91 days incubation. These observations suggest that community five could be utilised for the detoxification of PAH-contaminated soil.     

Degradation of polycyclic aromatic hydrocarbons in soil by a two-step sequential treatment

The objectives of this work were to isolate the microorganisms responsible for a previously observed degradation of polycyclic aromatic hydrocarbons (PAH) in soil and to test a method for cleaning a PAH-contaminated soil. An efficient PAH degrader was isolated from an agricultural soil and designated as Mycobacterium LP1. In liquid culture, it degraded phenanthrene (58%), pyrene (24%), anthracene (21%) and benzo(a)pyrene (10%) present in mixture (initial concentration 50 μg ml⁻¹ each) and phenanthrene (92%) and pyrene (94%) as sole carbon sources after 14 days of incubation at 30°C. In soil, Mycobacterium LP1 mineralised ¹⁴C-phenanthrene (45%) and ¹⁴C-pyrene (65%) after 10 days. The good ability of this Mycobacterium was combined with the benzo(a)pyrene oxidation effect obtained by 1% w/w rapeseed oil in a sequential treatment of a PAH-spiked soil (total PAH concentration 200 mg kg⁻¹). The first step was incubation with the bacterium for 12 days and the second step was the addition of the rapeseed oil after this time and a further incubation of 22 days. Phenanthrene (99%), pyrene (95%) and anthracene (99%) were mainly degraded in the first 12 days and a total of 85% of benzo(a)pyrene was transformed during the whole process. The feasibility of the method is discussed.     

PAH Degradation Capacity of Soil Microbial Communities—Does It Depend on PAH Exposure?

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of the environment. But is their microbial degradation equally wide in distribution? We estimated the PAH degradation capacity of 13 soils ranging from pristine locations (total PAHs ≈ 0.1 mg kg^?1) to heavily polluted industrial sites (total PAHs ≈ 400 mg kg^?1). The size of the pyrene- and phenanthrene-degrading bacterial populations was determined by most probable number (MPN) enumeration. Densities of phenanthrene degraders reflected previous PAH exposure, whereas pyrene degraders were detected only in the most polluted soils. The potentials for phenanthrene and pyrene degradation were measured as the mineralization of ¹⁴C-labeled spikes. The time to 10% mineralization of added ¹⁴C phenanthrene and ¹⁴C pyrene was inversely correlated with the PAH content of the soils. Substantial ¹⁴C phenanthrene mineralization in all soils tested, including seven unpolluted soils, demonstrated that phenanthrene is not a suitable model compound for predicting PAH degradation in soils. ¹⁴C pyrene was mineralized by all Danish soil samples tested, regardless of whether they were from contaminated sites or not, suggesting that in industrialized areas the background level of pyrene is sufficient to maintain pyrene degradation traits in the gene pool of soil microorganisms. In contrast, two pristine forest soils from northern Norway and Ghana mineralized little ¹⁴C pyrene within the 140-day test period. Mineralization of phenanthrene and pyrene by all Danish soils suggests that soil microbial communities of inhabited areas possess a sufficiently high PAH degradation capacity to question the value of bioaugmentation with specific PAH degraders for bioremediation.     

Phytoremediation and removal mechanisms in Bouteloua curtipendula growing in sterile hydrocarbon spiked cultures

Tolerance index and phytoremediation factors of side oats grama (Bouteloua curtipendula) with recalcitrant polycyclic aromatic hydrocarbons (PAH) phenanthrene (PHE), pyrene (PYR), and benzo[a]pyrene (BaP) and the resulting impact on phenotypic response, were evaluated in sterile conditions with whole plant growing in test-tube cultures with MS medium with PAH and compared with Tall fescue (Festuca arundinacea), control for this study. PAH mixture of PHE, PYR and BaP (1:1:1 w/w/w) blended with Maya crude oil (1:1 w/w), final concentration of 1500 mg kg(-1) was used. After 40 days, BaP removal, in the presence of Maya crude was superior compared with PHE and PYR removal Although the presence of PAH negatively affects the phenotypic response of the plants; sterile conditions experiments were helpful to evaluate phytoremediation factors to elucidate some important questions regarding phytoremediation mechanisms; in this study, B. curtipendula was able to phytostabilizate BaP associated to a significant hydrocarbon removal (57.4%) with high root accumulation but attenuated transport to stems, here reported as translocation factor. To our knowledge, this is the first time that quantifiable phytoremediation factors were used to evaluate the tolerance and removal capacity of a native semi-arid climate plant which is probably able to phytoremediate hydrocarbon contaminated soils.     

The Halophyte Cakile maritima Reduces Phenanthrene Phytotoxicity

In a previous study, we showed that the halophyte plant model Thellungiella salsuginea was more tolerant to phenanthrene (Polycyclic Aromatic Hydrocarbon: PAH) than its relative glycophyte Arabidopsis thaliana. In the present work, we investigated the potential of another halophyte with higher biomass production, Cakile maritma, to reduce phenanthrene phytotoxicity. Sand was used instead of arable soil with the aim to avoid pollutant degradation by microorganisms or their interaction with the plant. After 6 weeks of treatment by 500 ppm phenanthrene (Phe), stressed plants showed a severe reduction (–73%) in their whole biomass, roots being more affected than leaves and stems. In parallel, Guaiacol peroxidase (GPX) activity was increased by 185 and 62% in leaves and roots, respectively. Non-enzymatic antioxidant capacity (assayed by ABTS test) was maintained unchanged in all plant organs. The model halophytic plant Thellungiella salsuginea was used as a biomarker of phenanthrene stress severity and was grown at 0 (control), 125, 250, and 375 ppm. T. salsuginea plants grown on the sand previously contaminated by 500 ppm Phe then treated by C. maritma culture (phytoremediation culture) showed similar biomass production as plants subjected to 125 ppm Phe. This suggests that the phytotoxic effects of phenanthrene were reduced by 75% by the 6-week treatment by C. maritima. Our findings indicate that C. maritima can constitute a potentially good candidate for PAH phytoremediation.     

Inoculation of seed-borne fungus in the rhizosphere of Festuca arundinacea promotes hydrocarbon removal and pyrene accumulation in roots

Background and aims

The selective inoculation of specific hydrocarbon-degrading microbes into the plant rhizosphere offers a useful means for remediating hydrocarbon-contaminated soils. The effect of inoculating a seed-borne filamentous fungus (Lewia sp.) on hydrocarbon removal by Festuca arundinacea and its growth was studied on perlite (model soil) and soil, both spiked with hydrocarbons.

Methods

A hydrocarbon mixture (1,500 mg kg^?1) of two polycyclic aromatic hydrocarbons (PAH), phenanthrene and pyrene, blended with hexadecane (1.0:0.5:0.5 weight) was used. Greenhouse experiments were carried out for 45 days. Inoculated and non-inoculated plants were grown in dark cylindrical glass pots containing perlite or soil.

Results

Inoculation with Lewia sp. stimulated (100 %) root growth in spiked perlite. Inoculated plants showed higher phenanthrene removal (100 %) compared to non-inoculated plants in perlite and soil. Pyrene removal by inoculated plants was 37-fold higher than that by non-inoculated plants in perlite; in soil, pyrene removal by inoculated plants (97.9 %) differed significantly from that of non-inoculated plants (91.4 %). Accumulation of pyrene in roots (530.9 mg kg^?1 of dry roots) was promoted in perlite.

Conclusions

Our results demonstrate that Lewia sp. (endophytic fungus) improved the efficiency of PAH removal by F. arundinacea, on both perlite and soil, stimulating pyrene accumulation in roots.     

Inoculation of PAH-degrading strains of Fusarium solani and Arthrobacter oxydans in rhizospheric sand and soil microcosms: microbial interactions and PAH dissipation

Very little is known about the influence of bacterial-fungal ecological interactions on polycyclic aromatic hydrocarbon (PAH) dissipation in soils. Fusarium solani MM1 and Arthrobacter oxydans MsHM11 can dissipate PAHs in vitro. We investigated their interactions and their effect on the dissipation of three PAHs—phenanthrene (PHE), pyrene (PYR) and dibenz(a,h)anthracene (DBA)—in planted microcosms, in sterile sand or non-sterile soil. In sterile sand microcosms planted with alfalfa, the two microbes survived and grew, without any significant effect of co-inoculation. Co-inoculation led to the dissipation of 46 % of PHE after 21 days. In soil microcosms, whether planted with alfalfa or not, both strains persisted throughout the 46 days of the experiment, without any effect of co-inoculation or of alfalfa, as assessed by real-time PCR targeting taxon-level indicators, i.e. Actinobacteria 16S rDNA and the intergenic transcribed spacer specific to the genus Fusarium. The microbial community was analyzed by temporal temperature gradient electrophoresis and real-time PCR targeting bacterial and fungal rDNA and PAH-ring hydroxylating dioxygenase genes. These communities were modified by PAH pollution, which selected PAH-degrading bacteria, by the presence of alfalfa and, concerning the bacterial community, by inoculation. PHE and PYR concentrations significantly decreased (91 and 46 %, respectively) whatever the treatment, but DBA concentration significantly decreased (30 %) in planted and co-inoculated microcosms only.     

Characterization of a Polycyclic Aromatic Hydrocarbon-Degrading Microbial Consortium from a Petrochemical Sludge Landfarming Site

Anthracene, phenanthrene, and pyrene are polycyclic aromatic hydrocarbon (PAHs) that display both mutagenic and carcinogenic properties. They are recalcitrant to microbial degradation in soil and water due to their complex molecular structure and low solubility in water. This study presents the characterization of an efficient PAH (anthracene, phenanthrene, and pyrene)-degrading microbial consortium, isolated from a petrochemical sludge landfarming site. Soil samples collected at the landfarming area were used as inoculum in Warburg flasks containing soil spiked with 250 mg kg^-1 of anthracene. The soil sample with the highest production of CO₂-C in 176 days was used in liquid mineral medium for further enrichment of anthracene degraders. The microbial consortium degraded 48%, 67%, and 22% of the anthracene, phenanthrene, and pyrene in the mineral medium, respectively, after 30 days of incubation. Six bacteria, identified by 16S rRNA sequencing as Mycobacterium fortuitum, Bacillus cereus, Microbacterium sp., Gordonia polyisoprenivorans, two Microbacteriaceae bacteria, and a fungus identified as Fusarium oxysporum were isolated from the enrichment culture. The consortium and its monoculture isolates utilized a variety of hydrocarbons including PAHs (pyrene, anthracene, phenanthrene, and naftalene), monoaromatics hydrocarbons (benzene, ethylbenzene, toluene, and xylene), aliphatic hydrocarbons (1-decene, 1-octene, and hexane), hydrocarbon mixtures (gasoline and diesel oil), intermediary metabolites of PAHs degradation (catechol, gentisic acid, salicylic acid, and dihydroxybenzoic acid) and ethanol for growth. Biosurfactant production by the isolates was assessed by an emulsification index and reduction of the surface tension in the mineral medium. Significant emulsification was observed with the isolates, indicating production of high-molecular-weigh surfactants. The high PAH degradation rates, the wide spectrum of hydrocarbons utilization, and emulsification capacities of the microbial consortium and its member microbes indicate that they can be used for biotreatment and bioaugumentation of soils contaminated with PAHs.     

Isolation and genetic identification of PAH degrading bacteria from a microbial consortium

Polycyclic aromatic hydrocarbons (PAH; naphthalene, anthracene and phenanthrene) degrading microbial consortium C2PL05 was obtained from a sandy soil chronically exposed to petroleum products, collected from a petrochemical complex in Puertollano (Ciudad Real, Spain). The consortium C2PL05 was highly efficient degrading completely naphthalene, phenanthrene and anthracene in around 18 days of cultivation. The toxicity (Microtox™ method) generated by the PAH and by the intermediate metabolites was reduced to levels close to non-toxic in almost 40 days of cultivation. The identified bacteria from the contaminated soil belonged to γ-proteobacteria and could be include in Enterobacter and Pseudomonas genus. DGGE analysis revealed uncultured Stenotrophomonas ribotypes as a possible PAH degrader in the microbial consortium. The present work shows the potential use of these microorganisms and the total consortium for the bioremediation of PAH polluted areas since the biodegradation of these chemicals takes place along with a significant decrease in toxicity.     

Mycoremediation of PAH-contaminated soil

Out of a number of white-rot fungal cultures, strains ofIrpex lacteus andPleurotus ostreatus were selected for degradation of 7 three- and four-ring unsubstituted aromatic hydrocarbons (PAH) in two contaminated industrial soils. Respective data for removal of PAH in the two industrial soils byI. lacteus were: fluorene (41 and 67%), phenanthrene (20 and 56%), anthracene (29 and 49%), fluoranthene (29 and 57%), pyrene (24 and 42%), chrysene (16 and 32%) and benzo[a]anthracene (13 and 20%). In the same two industrial soilsP. ostreatus degraded the PAH with respective removal figures of fluorene (26 and 35%), phenanthrene (0 and 20%), anthracene (19 and 53%), fluoranthene (29 and 31%), pyrene (22 and 42%), chrysene (0 and 42%) and benzo[a]anthracene (0 and 13%). The degradation of PAH was determined against concentration of PAH in non-treated contaminated soils after 14 weeks of incubation. The fungal degradation of PAH in soil was studied simultaneously with ecotoxicity evaluation of fungal treated and non-treated contaminated soils. Compared to non-treated contaminated soil, fungus-treated soil samples indicated decrease in inhibition of bioluminescence in luminescent bacteria (Vibrio fischerii) and increase in germinated mustard (Brassica alba) seeds. An erratum to this article is available at .     

Pilot Scale Bioremediation of Creosote-Contaminated Soil—Efficacy of Enhanced Natural Attenuation and Bioaugmentation Strategies

In this study, the efficacy of bioremediation strategies (enhanced natural attenuation with nitrate and phosphate addition [ENA] and bioaugmentation) for the remediation of creosote-contaminated soil (7767 ± 1286 mg kg^?1 of the 16 EPA priority PAHs) was investigated at pilot scale. Bioaugmentation of creosote-contaminated soil with freshly grown or freeze dried Mycobacterium sp. strain 1B (a PAH degrading microorganism) was applied following bench scale studies that indicated that the indigenous soil microflora had a limited PAH metabolic activity. After 182 days, the total PAH concentration in creosote-contaminated soil was reduced from 7767 ± 1286 mg kg^?1 to 5579 ± 321 mg kg^?1, 2250 ± 71 mg kg^?1, 2050 ± 354 mg kg^?1 and 1950 ± 70 mg kg^?1 in natural attenuation (no additions) and ENA biopiles and biopiles augmented with freshly grown or freeze dried Mycobacterium sp. strain 1B respectively. In ENA and bioaugmentation biopiles, between 82% and 99% of three-ring compounds (acenaphthene, anthracene, fluorene, phenanthrene) were removed while four-ring PAH removal ranged from 33 to 81%. However, the extent of PAH degradation did not vary significantly between the ENA treatment and biopiles augmented with Mycobacterium sp. strain 1B. Four-ring PAH removal followed the order fluoranthene > pyrene > benz[a]anthracene > chrysene. The high residual concentration of some four-ring PAHs may be attributable to bioavailability issues rather than a lack of microbial catabolic activity. Comparable results between ENA and bioaugmentation at pilot scale were surprising given the limited degradative capacity of the microbial consortia enriched from the creosote-contaminated soil.     

Effect of rapeseed oil on the degradation of polycyclic aromatic hydrocarbons in soil by Rhodococcus wratislaviensis

The effect of rapeseed oil (0, 0.1 and 1% w/w) on the degradation of polycyclic aromatic hydrocarbons (PAH) by Rhodococcus wratislaviensis was studied in soils artificially contaminated with phenanthrene, anthracene, pyrene and benzo(a)pyrene (50 mg kg⁻¹ each), during 49 days at 30 °C. Without or with 0.1% of rapeseed oil, R. wratislaviensis degraded >90% of phenanthrene and anthracene in 14 days and mineralised approx. 23% of ¹⁴C-phenanthrene. The native microflora degraded pyrene (90% degradation; 75% mineralisation) and benzo(a)pyrene (30% degradation, no mineralisation). With 1% rapeseed oil, R. wratislaviensis degraded only 66% of the phenanthrene and mineralised 12.4%, and had no effect on other PAH, while degradation by the native microflora was inhibited. On the other hand, the addition of 1% oil promoted degradation of benzo(a)pyrene (75%) and anthracene (90%) and anthraquinone was produced at high concentrations and accumulated. Two distinct processes gave degradation of PAH, one biological and one abiotic. Biological processes mainly degraded phenanthrene and pyrene, either by R. wratislaviensis or by the indigenous microflora. Benzo(a)pyrene was degraded mainly by an abiotic process in the presence of 1% rapeseed oil. Anthracene was degraded by a combination of both processes.PAH are often found in contaminated soils and there is the need of developing techniques that can be applied in the remediation of these sites, where PAH, specially those with high molecular weight, pose health and environmental risks. There is a continuous search for efficient microorganisms able to degrade these pollutants and for methods to enhance their degradation and bioavailability, e.g. by the use of vegetable oils. This paper presents a novel process for the degradation of PAH by a combined biological/abiotic system.     

Utilizing pyrene-degrading endophytic bacteria to reduce the risk of plant pyrene contamination

Aims

Endophytic bacteria are ubiquitous in plants, but little information is available on the influence of endophytic bacteria on the uptake and metabolism of PAH by plants. Thus, we seek to investigate whether the colonization of a target plant by a PAH-degrading endophytic bacterium would improve the PAH metabolism of the plant and reduce the risk of plant PAH contamination.

Methods

A pyrene-degrading endophyte was isolated from PAH-contaminated plants using enrichment culture. After root inoculation with the isolated bacterium, greenhouse container experiments were conducted. Pyrene residues in soil and plant samples were analyzed by HPLC.

Results

A pyrene-degrading endophytic bacterium, Staphylococcus sp. BJ06, was isolated from Alopecurus aequalis and could degrade 56.0 % of pyrene (50 mg?·?L^?1) within 15 days. BJ06 grew and degraded pyrene efficiently under environmental conditions. The bacterium significantly promoted ryegrass growth and pyrene removal from contaminated soil in container experiments. The pyrene concentrations in ryegrass roots and shoots in endophyte-inoculated planted soil were reduced by 31.01 % and 44.22 %, respectively, compared with endophyte-free planted soil.

Conclusions

We have provided new perspectives on the regulation and control of plant uptake of organic contaminants with endophytic bacteria. The results of this study will be valuable to risk assessments of plant PAH contamination.     

Changes in phytotoxicity of polycyclic aromatic hydrocarbons in the course of microbial degradation

Phytotoxicity of six polycyclic aromatic hydrocarbons (PAHs) and their 16 oxidized derivatives that may be microbial metabolites arising in the course of PAH degradation was determined using an express test with the seedlings of sorghum (Sorghum bicolor L. Moench) and alfalfa (Medicago sativa L.). It was shown that germinating capacity is the least informative characteristic and the most useful parameter is development of seedlings during 3 days in the presence of compounds under investigation. Among unsubstituted compounds, toxicity in respect to seedlings decreased in the series fluorene > phenanthrene > anthracene. Chrysene, fluoranthene, and pyrene stimulated shoot development. It was found that some of the metabolites produced as a result of microbial degradation of phenanthrene (9,10-phenanthrenequinone, 1-hydroxy-2-naphthoic and benzoic acids) are more toxic for plants than starting PAH molecules. The obtained results are important for understanding rhizosphere processes associated with phytoremediation technique.     

Microbiological characterization of a coke oven contaminated site and evaluation of its potential for bioremediation

The soil microbial population of a coke oven site was investigated in order to evaluate its potential for bioremediation. The study was carried out in soil samples with distinct polynuclear aromatic hydrocarbon (PAH) contamination levels, comparing the population profiles constituted by total heterotrophic and PAH-utilizing strains. Isolation of degrading strains was performed with phenanthrene or pyrene as sole carbon sources. The ability to degrade other PAHs, such as anthracene, fluorene and fluoranthene was also investigated. The results showed a reduction of 30% in species diversity and microbial density drops one order of magnitude in contaminated samples. Furthermore, the number of PAH-utilizing colonies was higher in the contaminated area and about 20% of the isolates were able to degrade phenanthrene and pyrene, while this value decreased to 0.15% in uncontaminated samples. Three PAH-degrader strains were identified as: CDC gr. IV C-2, Aeromonas sp. and Pseudomonas vesicularis. The ability of these strains to degrade other PAHs was also investigated.     

Predicting the Efficacy of Polycyclic Aromatic Hydrocarbon Bioremediation in Creosote-Contaminated Soil Using Bioavailability Assays

Nonexhaustive extraction (propanol, butanol, hydroxypropyl-β-cyclodextrin [HPCD]), persulfate oxidation and biodegradability assays were employed to determine the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soil. After 16 weeks incubation, greater than 89% of three-ring compounds (acenaphthene, anthracene, fluorene, and phenanthrene) and 21% to 79% of four-ring compounds (benz[a]anthracene, chrysene, fluoranthene, and pyrene) were degraded by the indigenous microorganisms under biopile conditions. No significant decrease in five- (benzo[a]pyrene, benzo[b+k]fluoranthene) and six-ring compounds (benz[g,h,i]perylene, indeno[1,2,3-c,d]pyrene) was observed. Desorption of PAHs using propanol or butanol could not predict PAH biodegradability: low-molecular-weight PAH biodegradability was underestimated whereas high-molecular-weight PAH biodegradability was overestimated. Persulfate oxidation and HPCD extraction of creosote-contaminated soil was able to predict three- and four-ring PAH biodegradability; however, the biodegradability of five-ring PAHs was overestimated. These results demonstrate that persulfate oxidation and HPCD extraction are good predictors of PAH biodegradability for compounds with octanol-water partitioning coefficients of < 6.     

Bioremediation of a polyaromatic hydrocarbon contaminated soil by native soil microbiota and bioaugmentation with isolated microbial consortia

Biodegradation of a mixture of PAHs was assessed in forest soil microcosms performed either without or with bioaugmentation using individual fungi and bacterial and a fungal consortia. Respiratory activity, metabolic intermediates and extent of PAH degradation were determined. In all microcosms the low molecular weight PAH’s naphthalene, phenanthrene and anthracene, showed a rapid initial rate of removal. However, bioaugmentation did not significantly affect the biodegradation efficiency for these compounds. Significantly slower degradation rates were demonstrated for the high molecular weight PAH’s pyrene, benz[a]anthracene and benz[a]pyrene. Bioaugmentation did not improve the rate or extent of PAH degradation, except in the case of Aspergillus sp. Respiratory activity was determined by CO₂ evolution and correlated roughly with the rate and timing of PAH removal. This indicated that the PAHs were being used as an energy source. The native microbiota responded rapidly to the addition of the PAHs and demonstrated the ability to degrade all of the PAHs added to the soil, indicating their ability to remediate PAH-contaminated soils.     

Degradation of polycyclic aromatic hydrocarbons by an immobilized mixed bacterial culture

The mixed bacterial culture MK1 was capable of degrading a wide spectrum of aromatic compounds both as free and as immobilized cells. By offering anthracene oil or a defined mixture of phenol, naphthalene, phenanthrene, anthracene and pyrene (in concentrations of 0.1–0.2 mm, respectively) as sources of carbon and energy, a specific degradation pattern correlating with the condensation degree was observed. Regarding the defined mixture of aromatic hydrocarbons, complete metabolism was reached for phenol (0.1 mm) after 1 day, for naphthalene (0.1 mm) after 2 days and for phenanthrene (0.1 mm) after 15 days of cultivation. The conversion of anthracene (0.1 mm) and pyrene (0.1 mm) resulted in minimal residual concentrations, analogous to fluoranthene and pyrene of the anthracene oil (0.1%). Maximal total degradation for the tricyclic compounds dibenzofurane, fluorene, dibenzothiophene, phenanthrene and anthracene of the anthracene oil (0.1%) occurred after 5 days. In general, a significant metabolisation of the tetracyclic aromatic hydrocarbons fluoranthene and pyrene was observed after the degradation of phenol, naphthalene and most of the tricyclic compounds. Doubling the start concentrations of the polycyclic aromatic hydrocarbons effected higher degradation rates. Cell growth occurred simultaneously with the conversion of phenol, naphthalene and the tricyclic compounds. The immobilized cells showed stable growth and, compared to freely suspended cells, the same degradation sequence as well as an equivalent degradation potential — even in a model soil system. Correspondence to: I. Wiesel