Isolation of PAH-degrading bacteria from mangrove sediments and their biodegradation potential

Surface sediment samples were collected from seven mangrove swamps in Hong Kong SAR with different degrees of contamination. The total concentrations of 16 PAHs in these sediments ranged from 169.41 to 1058.37 ng g⁻¹ with the highest concentration found in Ma Wan and the lowest in Kei Ling Ha Lo Wai mangrove swamp. In each swamp, three bacterial consortia were enriched from sediments using phenanthrene (Phe) as the sole carbon and energy source, and individual bacterial colony showing Phe degradation was isolated and identified by 16S rDNA gene sequence. The consortia enriched from Sai Keng and Ho Chung sediments had highest ability to degrade mixed PAHs in liquid medium, with 90% Phe and Fla (fluoranthene) degraded in 7 days. On the other hand, Kei Ling Ha Lo Wai-enriched consortia degraded less than 40% Phe and Fla. Pyrene (Pyr) was hardly degraded by the consortia enriched from sediments. Bacterial isolates, namely Rhodococcus (HCCS), Sphingomonas (MWFG) and Paracoccus (SPNT) were capable to degrade mixed PAHs (Phe + Fla + Pyr). Their degradation percentages could be lower, comparable or even higher than their respective enriched consortia, depending on the consortium and the type of PAH compounds. These results suggest that PAH-degrading bacteria enriched from mangrove sediments, either as a mixed culture or as a single isolate could be used for PAHs bioremediation.     

Natural attenuation, biostimulation and bioaugmentation on biodegradation of polycyclic aromatic hydrocarbons (PAHs) in mangrove sediments

The biodegradability of a mixture of PAHs, namely fluorene (Fl), phenanthrene (Phe) and pyrene (Pyr), in mangrove sediment slurry was investigated. At the end of week 4, natural attenuation based on the presence of autochthonous microorganisms degraded more than 99% Fl and Phe but only around 30% of Pyr were degraded. Biostimulation with addition of mineral salt medium degraded over 97% of all three PAHs, showing that nutrient amendment could enhance Pyr degradation. Bioaugmentation with inoculation of a PAH-degrading bacterial consortium enriched from mangrove sediments did not show any promotion effect and the degradation percentages of three PAHs were similar to that by natural attenuation. Some inhibitory effect was observed in bioaugmentation treatment in week 1 with only 50% Fl and 70% Phe degraded. These results indicate that autochthonous microbes may interact and even compete with the enriched consortium during PAH biodegradation. Natural attenuation appeared to be the most appropriate way to remedy Fl- and Phe-contaminated mangrove sediments while biostimulation was more capable to degrade Pyr-contaminated sediments. The study also shows that although a large portion of the added PAHs (more than 95%) was adsorbed onto the sediments at the beginning of the experiment, most PAHs were degraded in 4 weeks, suggesting that the degraders could utilize the adsorbed PAHs efficiently.     

Differential gene expression in mummichogs (Fundulus heteroclitus) following treatment with pyrene: comparison to a creosote contaminated site

Mummichogs, Fundulus heteroclitus, an estuarine fish with a relatively small home range found along the eastern coast of the United States are well-suited to monitoring contaminant effects, including those of polycyclic aromatic hydrocarbons (PAHs). One of the common PAHs in estuaries is pyrene. We report here on efforts to develop multiple biomarkers of pyrene exposure in this species. Adult male mummichogs were exposed in the laboratory to the weak aryl hydrocarbon receptor (AhR) agonist pyrene at 0, 30, or 50 microg/L in 7-day static renewal exposures. The RNA was extracted from livers and alterations in mRNA expression were assessed by subtractive hybridization and differential display in order to produce multiple biomarkers of pyrene exposure. Genes demonstrating differential expression were confirmed by quantitative-PCR (Q-PCR) and include cytochrome P-450 1A (CYP1A), a putative hepatocyte growth factor activator, a X-ray inducible retrotransposon, and several expressed sequenced tags (ESTs). Some of these genes represent new biomarkers of pyrene exposure and potential biomarkers of PAH exposure. Therefore, similar changes were investigated at a Superfund site in Charleston, SC. Mummichogs from a creosote contaminated site and from a reference site (North Inlet National Estuarine Research Reserve near Georgetown, SC) were trapped, RNA extracted from the livers, and Q-PCR performed. Many of the genes differentially expressed following pyrene exposure were not altered at the creosote contaminated site in comparison to the reference site. However, CYP1A and an EST were induced. CYP1A induction at Diesel Creek indicates that this population of fish does not demonstrate refractory CYP1A phenotypes observed at several sites with high levels of AhR agonists. Ultimately, we anticipate that the use of multiple biomarkers of PAH exposure will provide useful information on the potential effects of toxicants.     

Effects of Compost Addition on Pyrene Removal from Soil Cultivated with Three Selected Plant Species

A greenhouse pot experiment was conducted to investigate the effect of compost addition on the phytoremediation ability of Medicago sativa, Brassica napus, and Lolium perenne in soils contaminated with pyrene. Pyrene concentrations were evaluated after 90 days in contaminated uncultivated amended‐soil, cultivated amended‐soils, and shoots and roots of the three plant species. The addition of compost enhances significantly pyrene dissipation from 16 to 26% in uncultivated soil, whereas in cultivated soils it appears not to have any significant effect on pyrene dissipation, neither pyrene was detectable in shoots and roots of the three species examined. The high partition coefficient of pyrene to compost dissolved organic matter (DOM) and the molar absorptivity values at 280 nm (ε₂₈₀) indicate a high affinity of pyrene to compost DOM molecules, likely due to their aromatic character. These results suggest that compost improves pyrene removal from soil, possibly by promoting its adsorption onto compost DOM. This property is very important in indicating that compost can be used, besides for its amendment capacity, also as a potential tool for remediation of contaminated soils.     

Abilities and genes for PAH biodegradation of bacteria isolated from mangrove sediments from the central of Thailand

PAH-degrading bacteria, including Novosphingobium sp. PCY, Microbacterium sp. BPW, Ralstonia sp. BPH, Alcaligenes sp. SSK1B, and Achromobacter sp. SSK4, were isolated from mangrove sediments. These isolates degraded 50–76% of 100 mg/l phenanthrene within 2 weeks. Strains PCY and BPW also degraded pyrene at 98% and 71%, respectively. Furthermore, all of them probably produced biosurfactants in the presence of hydrocarbons. Interestingly, PCY has a versatility to degrade various PAHs. Molecular techniques and plasmid curing remarkably revealed the presence of the alpha subunit of pyrene dioxygenase gene (nidA), involving in its pyrene/phenanthrene degrading ability, located on megaplasmid of PCY which has never before been reported in sphingomonads. Moreover, genes encoding ferredoxin, reductase, extradiol dioxygenase (bphA3A4C) and exopolysaccharide biosynthetase, which may be involved in PAH degradation and biosurfactant production, were also found in PCY. Therefore, we conclude that these isolates, especially PCY, can be the candidates for use as inoculums in the bioremediation.     

Stability and hydrogenation of polycyclic aromatic hydrocarbons during hydropyrolysis (HyPy) – Relevance for high maturity organic matter

A series of hydropyrolysis (HyPy) experiments have been conducted on a small suite of authentic polycyclic aromatic hydrocarbons (PAHs: coronene, pyrene and perylene) to investigate the HyPy behaviour of these PAHs. This information may help in the interpretation of the structural significance of aromatic HyPy products, often detected in high abundance, from high maturity kerogens. The PAHs were separately treated by HyPy and were all susceptible to some extent of hydrogenation. Perylene also decomposed into low molecular weight aromatics (e.g. methylbiphenyls). Structurally, perylene is much less stable than the more condensed PAHs coronene and pyrene. The total product concentrations (wt% of starting PAH) from all HyPy experiments were consistently less than 100 wt%, probably due to either the condensation of semi-volatile products on walls of the transfer line prior to reaching the HyPy trap or the inefficient cold trapping of highly volatile products. Hydrogenation of PAHs was prevalent and was found to be significantly influenced by the addition of a Mo-S based catalyst and potentially the C/Mo ratio, but largely independent of the two final temperatures used (520 °C and 550 °C). The fully aromatised and hydrogenated products for any stable ring system may provide a general indication of the size distribution of aromatic units within the kerogen structure.     

Pyrene induces a reduction in midbrain size and abnormal swimming behavior in early-hatched pufferfish larvae

Spills of heavy oil (HO) have an adverse effect on marine life. We have demonstrated previously that exposure to HO by fertilized eggs of the pufferfish (Takifugu rubripes) induces neural disruption and behavioral abnormality in early-hatched larvae. Here, two kinds of polycyclic aromatic hydrocarbons, pyrene and phenanthrene, were selected to examine their toxic effects on larval behavior of another pufferfish species (T. niphobles). Larvae exposed to pyrene or phenanthrene exhibited no abnormalities in morphology. However, those exposed to pyrene but not phenanthrene swam in an uncoordinated manner, although their swimming distance and speed were normal. The optic tectum, a part of the midbrain, of pyrene-exposed larvae did not grow to full size. Thus, these findings are indicated that pyrene might be a contributor to the behavioral and neuro-developmental toxicity, although there is no indication that it is the only compound participating in the toxicity of the heavy oil mixture.     

Marine-derived filamentous fungi and their potential application for polycyclic aromatic hydrocarbon bioremediation

Eight marine-derived fungi that were previously selected for their abilities to decolorize RBBR dye were subjected to pyrene and benzo[a]pyrene degradation. The fungus Aspergillus sclerotiorum CBMAI 849 showed the best performance with regard to pyrene (99.7%) and benzo[a]pyrene (76.6%) depletion after 8 and 16 days, respectively. Substantial amounts of benzo[a]pyrene (>50.0%) depletion were also achieved by Mucor racemosus CBMAI 847. Therefore, these two fungal strains were subjected to metabolism evaluation using the HPLC-DAD-MS technique. The results showed that A. sclerotiorum CBMAI 849 and M. racemosus CBMAI 847 were able to metabolize pyrene to the corresponding pyrenylsulfate and were able to metabolize benzo[a]pyrene to benzo[a]pyrenylsulfate, suggesting that the mechanism of hydroxylation is mediated by a cytochrome P-450 monooxygenase, followed by conjugation with sulfate ions. Because these fungi were adapted to the marine environment, the strains that were used in the present study are considered to be attractive targets for the bioremediation of saline environments, such as ocean and marine sediments that are contaminated by PAHs.     

Abnormalities in eelpout Zoarces viviparus upon chemical exposure

Elevated frequencies of abnormal embryos in female eelpout Zoarces viviparus have been demonstrated in Danish, Swedish and German monitoring programmes at certain geographic locations with high levels of anthropogenic input. Pollutants present in areas with high malformation frequencies were selected and tested in a controlled laboratory experiment for their potential to induce abnormalities among eelpout embryos upon injection into pregnant eelpout. Tributyltin, 2,3,7,8-tetrachlorodibenzo-p-dioxin, pyrene, nonylphenol, 2,2′,4,4′-tetrabromophenylether and heptadecafluorooctanesulfonic acid were tested, either individually or combined. Generally, the chemicals were transferred to eggs and/or embryos. Some of the exposures increased the proportion of broods with more than 10% abnormal or 5% malformed embryos, although the average percentages of abnormal development were not affected. Spinal, cranial and eye deformities were evident, similarly to what is seen in nature. Some of the exposures resulted in increased percentages of females with as well a low reproductive capacity as embryos with a low condition index.     

苯并(a)芘和芘对梭鱼肝脏谷胱甘肽过氧化酶活性的影响

在实验生态条件下，浓度范围0.1-50μg/L的苯并（a)芘和芘的短期暴露造成梭鱼肝脏GPx活性先抑制后诱导的效应，相同的污染物，高浓度组对GPx活性的抑制大于低浓度组，出现GPx活性诱导的时间早于低浓度组；相同浓度下，苯并（a）芘对GPx活的作用大于芘，这间接反映了苯并（a）芘和芘的毒性大小，这些结果说明梭鱼肝脏GPx活性与苯并（a）芘和芘暴露有一定的相关性，可以作为海洋环境污染监测的一种生物标志物。