The desert woodrat (Neotoma lepida) induces a diversity of biotransformation genes in response to creosote bush resin.

Liver biotransformation enzymes have long been thought to enable animals to feed on diets rich in xenobiotic compounds. However, despite decades of pharmacological research in humans and rodents, little is known about hepatic gene expression in specialized mammalian herbivores feeding on toxic diets. Leveraging a recently identified population of the desert woodrat (Neotoma lepida) found to be highly tolerant to toxic creosote bush (Larrea tridentata), we explored the expression changes of suites of biotransformation genes in response to diets enriched with varying amounts of creosote resin. Analysis of hepatic RNA-seq data indicated a dose-dependent response to these compounds, including the upregulation of several genes encoding transcription factors and numerous phase I, II, and III biotransformation families. Notably, elevated expression of five biotransformation families - carboxylesterases, cytochromes P450, aldo-keto reductases, epoxide hydrolases, and UDP-glucuronosyltransferases - corresponded to species-specific duplication events in the genome, suggesting that these genes play a prominent role in N. lepida's adaptation to creosote bush. Building on pharmaceutical studies in model rodents, we propose a hypothesis for how the differentially expressed genes are involved in the biotransformation of creosote xenobiotics. Our results provide some of the first details about how these processes likely operate in the liver of a specialized mammalian herbivore.

Application of Ozonation-Biodegradation Hybrid System for Polycyclic Aromatic Hydrocarbons Degradation.

Creosote, a mixture of polycyclic aromatic hydrocarbons (PAHs), was and is a wood impregnate of widespread use. Over the years the accumulation of creosote PAHs in soils and freshwaters has increased, causing a threat to ecosystems. The combined ozonation-biodegradation process is proposed to improve the slow and inefficient biodegradation of creosote hydrocarbons. The impact of different ozonation methods on the biodegradation of model wastewater was evaluated. The biodegradation rate, the changes in chemical oxygen demand, and the total organic carbon concentration were measured in order to provide insight into the process. Moreover, the bacteria consortium activity was monitored during the biodegradation step of the process. The collected data confirmed the research hypothesis, which was that the hybrid method can improve biodegradation. The pre-ozonation followed by inoculation with a bacteria consortium resulted in a significant increase in the biodegradation rate. It allows for the shortening of the time required for the consortium to reach maximum degradation effectiveness and cell activity. Hence, the study gives an important and useful perspective for the decontamination of creosote-polluted ecosystems.

PUF-Immobilized Bjerkandera adusta DSM 3375 as a Tool for Bioremediation of Creosote Oil Contaminated Soil.

Creosote oil, a byproduct of coal distillation, is primarily composed of aromatic compounds that are difficult to degrade, such as polycyclic aromatic hydrocarbons, phenolic compounds, and N-, S-, and O-heterocyclic compounds. Despite its toxicity and carcinogenicity, it is still often used to impregnate wood, which has a particularly negative impact on the condition of the soil in plants that impregnate wooden materials. Therefore, a rapid, effective, and eco-friendly technique for eliminating the creosote in this soil must be developed. The research focused on obtaining a preparation of Bjerkandera adusta DSM 3375 mycelium immobilized in polyurethane foam (PUF). It contained mold cells in the amount of 1.10 ± 0.09 g (DW)/g of the carrier. The obtained enzyme preparation was used in the bioremediation of soil contaminated with creosote (2% w/w). The results showed that applying the PUF-immobilized mycelium of B. adusta DSM 3375 over 5, 10, and 15 weeks of bioremediation, respectively, removed 19, 30, and 35% of creosote from the soil. After 15 weeks, a 73, 79, and 72% level of degradation of fluoranthene, pyrene, and fluorene, respectively, had occurred. The immobilized cells have the potential for large-scale study, since they can degrade creosote oil in soil.

Embryonic exposure to benzo[a]pyrene causes age-dependent behavioral alterations and long-term metabolic dysfunction in zebrafish.

Polycyclic aromatic hydrocarbons (PAH) are products of incomplete combustion which are ubiquitous pollutants and constituents of harmful mixtures such as tobacco smoke, petroleum and creosote. Animal studies have shown that these compounds exert developmental toxicity in multiple organ systems, including the nervous system. The relative persistence of or recovery from these effects across the lifespan remain poorly characterized. These studies tested for persistence of neurobehavioral effects in AB* zebrafish exposed 5-120 h post-fertilization to a typical PAH, benzo[a]pyrene (BAP). Study 1 evaluated the neurobehavioral effects of a wide concentration range of BAP (0.02-10 µM) exposures from 5 to 120 hpf during larval (6 days) and adult (6 months) stages of development, while study 2 evaluated neurobehavioral effects of BAP (0.3-3 µM) from 5 to 120 hpf across four stages of development: larval (6 days), adolescence (2.5 months), adulthood (8 months) and late adulthood (14 months). Embryonic BAP exposure caused minimal effects on larval motility, but did cause neurobehavioral changes at later points in life. Embryonic BAP exposure led to nonmonotonic effects on adolescent activity (0.3 µM hyperactive, Study 2), which attenuated with age, as well as startle responses (0.2 µM enhanced, Study 1) at 6 months of age. Similar startle changes were also detected in Study 2 (1.0 µM), though it was observed that the phenotype shifted from reduced pretap activity to enhanced posttap activity from 8 to 14 months of age. Changes in the avoidance (0.02-10 µM, Study 1) and approach (reduced, 0.3 µM, Study 2) of aversive/social cues were also detected, with the latter attenuating from 8 to 14 months of age. Fish from study 2 were maintained into aging (18 months) and evaluated for overall and tissue-specific oxygen consumption to determine whether metabolic processes in the brain and other target organs show altered function in late life based on embryonic PAH toxicity. BAP reduced whole animal oxygen consumption, and overall reductions in total basal, mitochondrial basal, and mitochondrial maximum respiration in target organs, including the brain, liver and heart. The present data show that embryonic BAP exposure can lead to neurobehavioral impairment across the life-span, but that these long-term risks differentially emerge or attenuate as development progresses.

Genome-wide scan reveals signatures of selection related to pollution adaptation in non-model estuarine Atlantic killifish (Fundulus heteroclitus).

In many human-altered ecosystems, organisms are increasingly faced with more diverse and complex environmental stressors and pollutant mixtures, to which the adaptations necessary to survive exposure are likely to be numerous and varied. Improving our understanding of the molecular mechanisms that underlie complex polygenic adaptations in natural settings requires significant toxicological, biochemical, physiological, and genomic data rarely available for non-model organisms. Here, we build upon two decades of study of adaptation to anthropogenic pollutants in a population of Atlantic killifish (Fundulus heteroclitus) that inhabits the creosote-contaminated Atlantic Wood Industries Superfund (AW) site on the Elizabeth River, Virginia in the United States. To better understand the genotypes that underlie previously characterized resistance to PCBs and PAHs, we performed Restriction site-Associated DNA sequencing (RADseq) on killifish from AW and two relatively clean reference sites (King's Creek-KC, and Mains Creek-MC). Across the genome, we analyzed over 83,000 loci and 12,000 single nucleotide polymorphisms (SNPs). Shared across both comparisons of killifish from polluted (AW) and relatively unpolluted (KC and MC) sites, we found eight genomic regions with smoothed FST values significantly (p < 0.001) elevated above background. Using the recently published F. heteroclitus reference genome, we identified candidate genes in these significant regions involved in the AHR pathway (e.g. AIP, ARNT1c), as well as genes relating to cardiac structure and function. These genes represent both previously characterized and potentially novel molecular adaptations involved with various aspects of resistance to these environmental toxins.

Biosurfactant-enhanced bioremediation of aged polycyclic aromatic hydrocarbons (PAHs) in creosote contaminated soil.

The potential for biological treatment of an environment contaminated by complex petrochemical contaminants was evaluated using creosote contaminated soil in ex situ bio-slurry reactors. The efficacy of biosurfactant application and stimulation of in situ biosurfactant production was investigated. The biosurfactant produced was purified and characterised using Fourier transform infrared (FTIR) spectroscopy. Biosurfactant enhanced degradation of PAHs was 86.5% (with addition of biosurfactant) and 57% in controls with no biosurfactant and nutrient amendments after incubation for 45 days. A slight decrease in degradation rate observed in the simultaneous biosurfactant and nutrient, NH4NO3 and KH2PO4, supplemented microcosm can be attributed to preferential microbial consumption of the biosurfactant supplemented. The overall removal of PAHs was determined to be mass transport limited since the dissolution rate caused by the biosurfactant enhanced the bioavailability of the PAHs to the microorganisms. The consortium culture was predominated by the aromatic ring-cleaving species Bacillus stratosphericus, Bacillus subtilis, Bacillus megaterium, and Pseudomonas aeruginosa.

Combination of biochar amendment and mycoremediation for polycyclic aromatic hydrocarbons immobilization and biodegradation in creosote-contaminated soil.

Soils impregnated with creosote contain high concentrations of polycyclic aromatic hydrocarbons (PAH). To bioremediate these soils and avoid PAH spread, different bioremediation strategies were tested, based on natural attenuation, biochar application, wheat straw biostimulation, Pleurotus ostreatus mycoremediation, and the novel sequential application of biochar for 21 days and P. ostreatus 21 days more. Soil was sampled after 21 and 42 days after the remediation application. The efficiency and effectiveness of each remediation treatment were assessed according to PAH degradation and immobilization, fungal and bacterial development, soil eco-toxicity and legal considerations. Natural attenuation and biochar treatments did not achieve adequate PAH removal and soil eco-toxicity reduction. Biostimulation showed the highest bacterial development but low PAH degradation rate. Mycoremediation achieved the best PAH degradation rate and the lowest bioavailable fraction and soil eco-toxicity. This bioremediation strategy achieved PAH concentrations below Spanish legislation for contaminated soils (RD 9/2005). Sequential application of biochar and P. ostreatus was the second treatment most effective for PAH biodegradation and immobilization. However, the activity of P. ostreatus was increased by previous biochar application and PAH degradation efficiency was increased. Therefore, the combined strategy for PAH degradation have high potential to increase remediation efficiency.

Evidence for functional convergence in genes upregulated by herbivores ingesting plant secondary compounds.

BACKGROUND: Nearly 40 years ago, Freeland and Janzen predicted that liver biotransformation enzymes dictated diet selection by herbivores. Despite decades of research on model species and humans, little is known about the biotransformation mechanisms used by mammalian herbivores to metabolize plant secondary compounds (PSCs). We investigated the independent evolution of PSC biotransformation mechanisms by capitalizing on a dramatic diet change event-the dietary inclusion of creosote bush (Larrea tridentata)-that occurred in the recent evolutionary history of two species of woodrats (Neotoma lepida and N. bryanti). RESULTS: By comparing gene expression profiles of two populations of woodrats with evolutionary experience to creosote and one population naïve to creosote, we identified genes either induced by a diet containing creosote PSCs or constitutively higher in populations with evolutionary experience of creosote. Although only one detoxification gene (an aldo-keto reductase) was induced by both experienced populations, these populations converged upon functionally equivalent strategies to biotransform the PSCs of creosote bush by constitutively expressing aldehyde and alcohol dehydrogenases, Cytochromes P450s, methyltransferases, glutathione S-transferases and sulfotransferases. The response of the naïve woodrat population to creosote bush was indicative of extreme physiological stress. CONCLUSIONS: The hepatic detoxification system of mammals is notoriously complex, with hundreds of known biotransformation enzymes. The comparison herein of woodrat taxa that differ in evolutionary and ecological experience with toxins in creosote bush reveals convergence in the overall strategies used by independent species after a historical shift in diet. In addition, remarkably few genes seemed to be important in this dietary shift. The research lays the requisite groundwork for future studies of specific biotransformation pathways used by woodrats to metabolize the toxins in creosote and the evolution of diet switching in woodrats. On a larger level, this work advances our understanding of the mechanisms used by mammalian herbivores to process toxic diets and illustrates the importance of the selective relationship of PSCs in shaping herbivore diversity.

Assessment of the efficiency of in situ bioremediation techniques in a creosote polluted soil: change in bacterial community.

This work aimed to assess the effectiveness of different in situ bioremediation treatments (bioaugmentation, biostimulation, bioaugmentation and biostimulation, and natural attenuation) on creosote polluted soil. Toxicity, microbial respiration, creosote degradation and the evolution of bacterial communities were analyzed. Results showed that creosote decreased significantly in all treatments, and no significant differences were found between treatments. However, some specific polycyclic aromatic hydrocarbons (PAH) were degraded to a greater extent by biostimulation. The dominance of low temperatures (8.9 °C average) slowed down microbial creosote and PAH uptake and, despite significantly creosote degradation (>60%) at the end of the experiment, toxicity remained constant and high throughout the biodegradation process. DGGE results revealed that biostimulation showed the highest microbial biodiversity, although at the end of the biodegradation process, community composition in all treatments was different from that of the control assay (unpolluted soil). The active uncultured bacteria belonged to the genera Pseudomonas, Sphingomonas, Flexibacter, Pantoea and Balneimonas, the latter two of which have not been previously described as PAH degraders. The majority of the species identified during the creosote biodegradation belonged to Pseudomonas genus, which has been widely studied in bioremediation processes. Results confirmed that some bacteria have an intrinsic capacity to degrade the creosote without previous exposure.

An integrated assessment of sediment remediation in a midwestern U.S. stream using sediment chemistry, water quality, bioassessment, and fish biomarkers.

A comprehensive biological, sediment, and water quality study of the lower Little Scioto River near Marion, Ohio, USA, was undertaken to evaluate the changes or improvements in biotic measurements following the removal of creosote-contaminated sediment. The study area covered 7.5 river miles (RMs), including a remediated section between RMs 6.0 and 6.8. Fish and macroinvertebrate assemblages, fish biomarkers (i.e., polycyclic aromatic hydrocarbon [PAH] metabolite levels in white sucker [Castostomus commersoni] and common carp [Cyprinus carpio] bile and DNA damage), sediment chemistry, and water quality were assessed at five locations relative to the primary source of historical PAH contamination-upstream (RM 9.2), adjacent (RM 6.5), and downstream (RMs 5.7, 4.4, and 2.7). Overall, the biomarker results were consistent with the sediment PAH results, showing a pattern of low levels of PAH bile metabolites and DNA damage at the upstream (reference or background location), as well as the remediated section, high levels at the two immediate downstream sites, and somewhat lower levels at the furthest downstream site. Results show that remediation was effective in reducing sediment contaminant concentrations and exposure of fish to PAHs and in improving fish assemblages (60% increase in index of biotic integrity scores) in remediated river sections. Additional remedial investigation and potentially further remediation is needed to improve the downstream benthic fish community, which is still heavily exposed to PAH contaminants.

Spatial uncoupling of biodegradation, soil respiration, and PAH concentration in a creosote contaminated soil.

Hotspots and coldspots of concentration and biodegradation of polycyclic aromatic hydrocarbons (PAHs) marginally overlapped at the 0.5-100 m scale in a creosote contaminated soil in southern Sweden, suggesting that concentration and biodegradation had little spatial co-variation. Biodegradation was substantial and its spatial variability considerable and highly irregular, but it had no spatial autocorrelation. The soil concentration of PAHs explained only 20-30% of the variance of their biodegradation. Soil respiration was spatially autocorrelated. The spatial uncoupling between biodegradation and soil respiration seemed to be governed by the aging of PAHs in the soil, since biodegradation of added 13C phenanthrene covaried with both soil respiration and microbial biomass. The latter two were also correlated with high concentrations of phospholipid fatty acids (PLFAs) that are common in gram-negative bacteria. However, several of the hotspots of biodegradation coincided with hotspots for the distribution of a PLFA indicative of fungal biomass.

Analysis of phenanthrene and benzo[a]pyrene tetraol enantiomers in human urine: relevance to the bay region diol epoxide hypothesis of benzo[a]pyrene carcinogenesis and to biomarker studies.

One widely accepted metabolic activation pathway of the prototypic carcinogenic polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) proceeds through the "bay region diol epoxide" BaP-(7R,8S)-diol-(9S,10R)-epoxide (2). However, few studies have addressed the analysis of human urinary metabolites of BaP, which result from this pathway. Phenanthrene (Phe) is structurally related to BaP, but human exposure to Phe is far greater, and its metabolites can be readily detected in urine. Thus, Phe metabolites have been proposed as biomarkers of PAH exposure and metabolic activation. Phe-tetraols in particular could be biomarkers of the diol epoxide pathway. While BaP-tetraols and Phe-tetraols have been previously quantified in human urine, no published studies have determined their enantiomeric composition. This is important because different enantiomers would result from the bay region diol epoxide and "reverse" diol epoxide pathways, the latter being associated with weak mutagenicity and carcinogenicity. We addressed this problem using chiral HPLC to separate the enantiomers of BaP-7,8,9,10-tetraol and Phe-1,2,3,4-tetraol. Urine samples from smokers were subjected to solid-phase extraction, chiral HPLC, and GC-NICI-MS/MS analysis for silylated Phe-1,2,3,4-tetraols. The results demonstrated that >96% of Phe-1,2,3,4-tetraol in smokers' urine was Phe-(1S,2R,3S,4R)-tetraol (12), resulting from the "reverse" diol epoxide pathway, whereas less than 4% resulted from the "bay region diol epoxide" pathway of Phe metabolism. Urine from creosote workers was similarly analyzed for BaP-7,8,9,10-tetraol enantiomers. In contrast to the results of the Phe-tetraol analyses, 78% of BaP-7,8,9,10-tetraol in these human urine samples was BaP-(7R,8S,9R,10S)-tetraol (3) resulting from the "bay region diol epoxide" pathway of BaP metabolism. These results provide further support for the bay region diol epoxide pathway of BaP metabolism in humans and demonstrate differences in BaP and Phe metabolism, which may be important when considering Phe-tetraols as biomarkers of PAH metabolic activation.

Functional genes reveal the intrinsic PAH biodegradation potential in creosote-contaminated groundwater following in situ biostimulation.

A small-scale functional gene array containing 15 functional gene probes targeting aliphatic and aromatic hydrocarbon biodegradation pathways was used to investigate the effect of a pilot-scale air sparging and nutrient infiltration treatment on hydrocarbon biodegradation in creosote-contaminated groundwater. Genes involved in the different phases of polycyclic aromatic hydrocarbon (PAH) biodegradation were detected with the functional gene array in the contaminant plume, thus indicating the presence of intrinsic biodegradation potential. However, the low aerobic fluorescein diacetate hydrolysis, the polymerase chain reaction (PCR) amplification of 16S rRNA genes closely similar to sulphate-reducing and denitrifying bacteria and the negligible decrease in contaminant concentrations showed that aerobic PAH biodegradation was limited in the anoxic groundwater. Increased abundance of PAH biodegradation genes was detected by functional gene array in the monitoring well located at the rear end of the biostimulated area, which indicated that air sparging and nutrient infiltration enhanced the intrinsic, aerobic PAH biodegradation. Furthermore, ten times higher naphthalene dioxygenase gene copy numbers were detected by real-time PCR in the biostimulated area, which was in good agreement with the functional gene array data. As a result, functional gene array analysis was demonstrated to provide a potential tool for evaluating the efficiency of the bioremediation treatment for enhancing hydrocarbon biodegradation in field-scale applications.

Microbial populations related to PAH biodegradation in an aged biostimulated creosote-contaminated soil.

A previous bioremediation survey on a creosote-contaminated soil showed that aeration and optimal humidity promoted depletion of three-ringed polycyclic aromatic hydrocarbons (PAHs), but residual concentrations of four-ringed benzo(a)anthracene (B(a)A) and chrysene (Chry) remained. In order to explain the lack of further degradation of heavier PAHs such as four-ringed PAHs and to analyze the microbial population responsible for PAH biodegradation, a chemical and microbial molecular approach was used. Using a slurry incubation strategy, soil in liquid mineral medium with and without additional B(a)A and Chry was found to contain a powerful PAH-degrading microbial community that eliminated 89% and 53% of the added B(a)A and Chry, respectively. It is hypothesized that the lack of PAH bioavailability hampered their further biodegradation in the unspiked soil. According to the results of the culture-dependent and independent techniques Mycobacterium parmense, Pseudomonas mexicana, and Sphingobacterials group could control B(a)A and Chry degradation in combination with several microorganisms with secondary metabolic activity.

Fungal bioremediation of the creosote-contaminated soil: influence of Pleurotus ostreatus and Irpex lacteus on polycyclic aromatic hydrocarbons removal and soil microbial community composition in the laboratory-scale study.

The aim of this study was to determine the efficacy of selected basidiomycetes in the removing of polycyclic aromatic hydrocarbons (PAH) from the creosote-contaminated soil. Fungi Pleurotus ostreatus and Irpex lacteus were supplemented with creosote-contaminated (50-200 mg kg(-1) PAH) soil originating from a wood-preserving plant and incubated at 15 °C for 120 d. Either fungus degraded PAH with 4-6 aromatic rings more efficiently than the microbial community present initially in the soil. PAH removal was higher in P. ostreatus treatments (55-67%) than in I. lacteus treatments (27-36%) in general. P. ostreatus (respectively, I. lacteus) removed 86-96% (47-59%) of 2-rings PAH, 63-72% (33-45%) of 3-rings PAH, 32-49% (9-14%) of 4-rings PAH and 31-38% (11-13%) of 5-6-rings PAH. MIS (Microbial Identification System) Sherlock analysis of the bacterial community determined the presence of dominant Gram-negative bacteria (G-) Pseudomonas in the inoculated soil before the application of fungi. Complex soil microbial community was characterized by phospholipid fatty acids analysis followed by GC-MS/MS. Either fungus induced the decrease of bacterial biomass (G- bacteria in particular), but the soil microbial community was influenced by P. ostreatus in a different way than by I. lacteus. The bacterial community was stressed more by the presence of I. lacteus than P. ostreatus (as proved by the ratio of the fungal/bacterial markers and by the ratio of trans/cis mono-unsaturated fatty acids). Moreover, P. ostreatus stimulated the growth of Gram-positive bacteria (G+), especially actinobacteria and these results indicate the potential of the positive synergistic interaction of this fungus and actinobacteria in creosote biodegradation.

"Pharm-ecology" of diet shifting: biotransformation of plant secondary compounds in creosote (Larrea tridentata) by a woodrat herbivore, Neotoma lepida.

Diet switching in mammalian herbivores may necessitate a change in the biotransformation enzymes used to process plant secondary compounds (PSCs). We investigated differences in the biotransformation system in the mammalian herbivore, Neotoma lepida, after a radical shift in diet and secondary compound composition. Populations of N. lepida in the Mojave Desert have evolved over the past 10,000 years to feed on creosote (Larrea tridentata) from an ancestral state of consuming juniper (Juniperus osteosperma). This dietary shift represents a marked change in the dietary composition of PSCs in that creosote leaves are coated with phenolic resin, whereas juniper is high in terpenes but lacks phenolic resin. We quantified the enzyme activity of five major groups of biotransformation enzymes (cytochrome P450s, NAD(P)H:quinone oxidoreductase, glutathione conjugation, sulfation, and glucuronidation) recognized for their importance to mammalian biotransformation for the elimination of foreign compounds. Enzyme activities were compared between populations of Mojave and Great Basin woodrats fed control and creosote diets. In response to creosote, the Mojave population had greater levels of cytochrome P450s (CYP2B, CYP1A) and glutathione conjugation liver enzymes compared with the Great Basin population. Our results suggest that elevated levels of cytochrome P450s and glutathione conjugation enzymes in the Mojave population may be the underlying biotransformation mechanisms that facilitate feeding on creosote.

Mycelium growth and degradation of creosote-treated wood by basydiomycetes.

Tolerance of wood decay fungi of the genera Agrocybe, Armillaria, Auricularia, Daedalea, Pleurotus, Trametes to the presence of various amounts of creosote-treated wood (CTW) in the growth medium was compared. In the case of the most tolerant strain, Pleurotus ostreatus SMR 684, extracellular laccase and peroxidase specific activities were monitored during growth in the presence of CTW. Degradation of various creosote-constituting polycyclic aromatic hydrocarbons by this strain was evaluated by GC-MS and the ecotoxicity of treated and untreated CTW was compared by Microtox test.

Simultaneous biodegradation of creosote-polycyclic aromatic hydrocarbons by a pyrene-degrading Mycobacterium.

When incubated with a creosote-polycyclic aromatic hydrocarbons (PAHs) mixture, the pyrene-degrading strain Mycobacterium sp. AP1 acted on three- and four-ring components, causing the simultaneous depletion of 25% of the total PAHs in 30 days. The kinetics of disappearance of individual PAHs was consistent with differences in aqueous solubility. During the incubation, a number of acid metabolites indicative of distinctive reactions carried out by high-molecular-weight PAH-degrading mycobacteria accumulated in the medium. Most of these metabolites were dicarboxylic aromatic acids formed as a result of the utilization of growth substrates (phenanthrene, pyrene, or fluoranthene) by multibranched pathways including meta- and ortho-ring-cleavage reactions: phthalic acid, naphthalene-1,8-dicarboxylic acid, phenanthrene-4,5-dicarboxylic acid, diphenic acid, Z-9-carboxymethylenefluorene-1-carboxylic acid, and 6,6'-dihydroxy-2,2'-biphenyl dicarboxylic acid. Others were dead-end products resulting from cometabolic oxidations on nongrowth substrates (fluorene meta-cleavage product). These results contribute to the general knowledge of the biochemical processes that determine the fate of the individual components of PAH mixtures in polluted soils. The identification of the partially oxidized compounds will facilitate to develop analytical methods to determine their potential formation and accumulation in contaminated sites.

Microbial community changes during the bioremediation of creosote-contaminated soil.

AIMS: To investigate the effects of aeration on the ex situ biodegradation of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soil and its effect on the microbial community present. METHODS AND RESULTS: Aerated and nonaerated microcosms of soil excavated from a former timber treatment yard were maintained and sampled for PAH concentration and microbial community changes by terminal restriction fragment length polymorphism (TRFLP) analysis. After an experimental period of just 13 days, degradation was observed with all the PAHs monitored. Abiotic controls showed no loss of PAH. Results unexpectedly showed greater loss of the higher molecular weight PAHs in the nonaerated control. This may have been due to the soil excavation causing initial decompaction and aeration and the resulting changes caused in the microbial community composition, indicated by TRFLP analysis showing several ribotypes greatly increasing in relative abundance. Similar changes in both microcosms were observed but with several possible key differences. The species of micro-organisms putatively identified included Bacilli, pseudomonad, aeromonad, Vibrio and Clostridia species. CONCLUSIONS: Excavation of the contaminated soil leads to decompaction, aeration and increased nutrient availability, which in turn allow microbial biodegradation of the PAHs and a change in the microbial community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the changes occurring in the microbial community during biodegradation of all PAHs is essential for the development of improved site remediation protocols. TRFLP allows useful monitoring of the total microbial community.

Effect of modified Fenton's reaction on microbial activity and removal of PAHs in creosote oil contaminated soil.

This study describes the removal of polycyclic aromatic hydrocarbons (PAHs) from creosote oil contaminated soil by modified Fenton's reaction in laboratory-scale column experiments and subsequent aerobic biodegradation of PAHs by indigenous bacteria during incubation of the soil. The effect of hydrogen peroxide addition for 4 and 10 days and saturation of soil with H(2)O(2) on was studied. In both experiments the H(2)O(2) dosage was 0.4 g H(2)O(2)/g soil. In completely H(2)O(2)-saturated soil the removal of PAHs (44% within 4 days) by modified Fenton reaction was uniform over the entire soil column. In non-uniformly saturated soil, PAH removal was higher in completely saturated soil (52% in 10 days) compared to partially saturated soil, with only 25% in 10 days. The effect of the modified Fenton's reaction on the microbial activity in the soil was assessed based on toxicity tests towards Vibrio fischeri, enumeration of viable and dead cells, microbial extracellular enzyme activity, and oxygen consumption and carbon dioxide production during soil incubation. During the laboratory-scale column experiments, the toxicity of column leachate towards Vibrio fischeri increased as a result of the modified Fenton's reaction. The activities of the microbial extracellular enzymes acetate- and acidic phosphomono-esterase were lower in the incubated modified Fenton's treated soil compared to extracellular enzyme activities in untreated soil. Abundance of viable cells was lower in incubated modified Fenton treated soil than in untreated soil. Incubation of soil in serum bottles at 20 degrees C resulted in consumption of oxygen and formation of carbon dioxide, indicating aerobic biodegradation of organic compounds. In untreated soil 20-30% of the PAHs were biodegraded during 2 months of incubation. Incubation of chemically treated soil slightly increased PAH-removal compared to PAH-removal in untreated soil.