Effects of Three Polycyclic Aromatic Hydrocarbons on Sediment Bacterial Community

Mangrove sediment is susceptible to anthropogenic pollutants, including polycyclic aromatic hydrocarbons (PAHs). However, the effects of PAHs on the bacterial diversity in mangrove sediment have been rarely studied. In the present study, the effects of three types of PAHs (Naphthalene, Fluorene, and Pyrene) at three doses on sediment microbial populations were investigated by using denaturing gradient gel electrophoresis (DGGE). After 7 and 24 days of incubation of the three types of PAHs, markedly different patterns were observed in the bacterial communities. Overall, the diversity of bacterial community was suppressed before 7 days but was promoted after 24 days. Multidimensional scaling analysis suggested that the composition of bacterial communities after 7 days was distinctly distant from that after 24 days. Also despite a slight shift of bacterial abundance, the bacterial communities were relatively steady in these sediments after exposure to PAHs. In addition, DGGE suggested that the applications of three PAHs (especially PYR) had considerable effects on bacterial communities. For phylogenetic analysis, bacteria species belonging to Proteobacteria (α-, β-, and γ-), Actinobacteria, Chloroflexi, Bacteroidetes, and Planctomycetes were changed dramatically after treatment with PAHs. These results suggest that PAHs play key roles in the change of bacterial community, which may be important for understanding the relationship between PAHs and sediment microbial ecology.     

Polycyclic Aromatic Hydrocarbon-Induced Signaling Events Relevant to Inflammation and Tumorigenesis in Lung Cells Are Dependent on Molecular Structure

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and occupational toxicants, which are a major human health concern in the U.S. and abroad. Previous research has focused on the genotoxic events caused by high molecular weight PAHs, but not on non-genotoxic events elicited by low molecular weight PAHs. We used an isomeric pair of low molecular weight PAHs, namely 1-Methylanthracene (1-MeA) and 2-Methylanthracene (2-MeA), in which only 1-MeA possessed a bay-like region, and hypothesized that 1-MeA, but not 2-MeA, would affect non-genotoxic endpoints relevant to tumor promotion in murine C10 lung cells, a non-tumorigenic type II alveolar pneumocyte and progenitor cell type of lung adenocarcinoma. The non-genotoxic endpoints assessed were dysregulation of gap junction intercellular communication function and changes in the major pulmonary connexin protein, connexin 43, using fluorescent redistribution and immunoblots, activation of mitogen activated protein kinases (MAPK) using phosphospecific MAPK antibodies for immunoblots, and induction of inflammatory genes using quantitative RT-PCR. 2-MeA had no effect on any of the endpoints, but 1-MeA dysregulated gap junctional communication in a dose and time dependent manner, reduced connexin 43 protein expression, and altered membrane localization. 1-MeA also activated ERK1/2 and p38 MAP kinases. Inflammatory genes, such as cyclooxygenase 2, and chemokine ligand 2 (macrophage chemoattractant 2), were also upregulated in response to 1-MeA only. These results indicate a possible structure-activity relationship of these low molecular weight PAHs relevant to non-genotoxic endpoints of the promoting aspects of cancer. Therefore, our novel findings may improve the ability to predict outcomes for future studies with additional toxicants and mixtures, identify novel targets for biomarkers and chemotherapeutics, and have possible implications for future risk assessment for these PAHs.     

Bench-Scale Phytoremediation of Polycyclic Aromatic Hydrocarbon-Contaminated Marine Sediment with Tropical Plants

Elevated levels of polycyclic aromatic hydrocarbons (PAHs) were found in dredged marine sediment from Pearl Harbor. The degradation of PAHs was investigated with soil-sediment systems (washed and unwashed) and 20 plant species. Marine sediment was diluted with native soil in the first experiment. Sediment was washed with gypsum solution and water in the second and third experiments, respectively, prior to soil dilution. Soil dilution ratios were 0, 6, 16, and 37% by weight in the first experiment, 0, 37, 64, and 100% in the second and 0, 18, 64, and 100% in the third. Seven tree, nine shrub, and four grass species were grown for 12 weeks in a glasshouse. A “no plant” control was used in all the experiments. Several plant species yielded up to 90% PAH degradation in the first experiment when compared with no plant control. In the second planting, dwarf hau (Hibiscus tiliaceus) and vetiver (Vetiver zizanoides) appeared to cause the greatest PAH reduction, while paspalum (Paspalum vaginatum) and naupaka (Scaevola sericea) did not. The greatest difference between control and planted soil for both benzo[a]pyrene and total PAH occurred at the highest sediment ratios.     

Diversity of ndo Genes in Mangrove Sediments Exposed to Different Sources of Polycyclic Aromatic Hydrocarbon Pollution

Polycyclic aromatic hydrocarbon (PAH) pollutants originating from oil spills and wood and fuel combustion are pollutants which are among the major threats to mangrove ecosystems. In this study, the composition and relative abundance in the sediment bacterial communities of naphthalene dioxygenase (ndo) genes which are important for bacterial adaptation to environmental PAH contamination were investigated. Three urban mangrove sites which had characteristic compositions and levels of PAH compounds in the sediments were selected. The diversity and relative abundance of ndo genes in total community DNA were assessed by a newly developed ndo denaturing gradient gel electrophoresis (DGGE) approach and by PCR amplification with primers targeting ndo genes with subsequent Southern blot hybridization analyses. Bacterial populations inhabiting sediments of urban mangroves under the impact of different sources of PAH contamination harbor distinct ndo genotypes. Sequencing of cloned ndo amplicons comigrating with dominant DGGE bands revealed new ndo genotypes. PCR-Southern blot analysis and ndo DGGE showed that the frequently studied nah and phn genotypes were not detected as dominant ndo types in the mangrove sediments. However, ndo genotypes related to nagAc-like genes were detected, but only in oil-contaminated mangrove sediments. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the abundance and diversity of ndo genes in sediments of urban mangroves.     

Action of a Fluoranthene-Utilizing Bacterial Community on Polycyclic Aromatic Hydrocarbon Components of Creosote

Cultures enriched by serial transfer through a mineral salts medium containing fluoranthene were used to establish a stable, seven-member bacterial community from a sandy soil highly contaminated with coal tar creosote. This community exhibited an ability to utilize fluoranthene as the sole carbon source for growth, as demonstrated by increases in protein concentration and changes in absorption spectra when grown on fluoranthene in liquid culture. Biotransformation of other polycyclic aromatic hydrocarbons (PAHs) was verified by demonstrating their disappearance from an artificial PAH mixture by capillary gas chromatography. When grown on fluoranthene as the sole carbon source and subsequently exposed to fluoranthene plus 16 additional PAHs typical of those found in creosote, this community transformed all PAHs present in this defined mixture. After 3 days of incubation, 13 of the original 17 PAH components were degraded to levels below the limit of detection (10 ng/liter). Continued incubation resulted in extensive degradation of the remaining four compounds. The ability of this community to utilize a high-molecular-weight PAH as the sole carbon source, in conjunction with its ability to transform a diverse array of PAHs, suggests that it may be of value in the bioremediation of environments contaminated with PAHs, such as those impacted by creosote.     

Bacterial Diversity of a Consortium Degrading High-Molecular-Weight Polycyclic Aromatic Hydrocarbons in a Two-Liquid Phase Biosystem

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. Although several PAH-degrading bacterial species have been isolated, it is not expected that a single isolate would exhibit the ability to degrade completely all PAHs. A consortium composed of different microorganisms can better achieve this. Two-liquid phase (TLP) culture systems have been developed to increase the bioavailability of poorly soluble substrates for uptake and biodegradation by microorganisms. By combining a silicone oil–water TLP system with a microbial consortium capable of degrading HMW PAHs, we previously developed a highly efficient PAH-degrading system. In this report, we characterized the bacterial diversity of the consortium with a combination of culture-dependent and culture-independent methods. Polymerase chain reaction (PCR) of part of the 16S ribosomal RNA gene (rDNA) sequences combined with denaturing gradient gel electrophoresis was used to monitor the bacterial population changes during PAH degradation of the consortium when pyrene, chrysene, and benzo[a]pyrene were provided together or separately in the TLP cultures. No substantial changes in bacterial profiles occurred during biodegradation of pyrene and chrysene in these cultures. However, the addition of the low-molecular-weight PAHs phenanthrene or naphthalene in the system favored one bacterial species related to Sphingobium yanoikuyae. Eleven bacterial strains were isolated from the consortium but, interestingly, only one—IAFILS9 affiliated to Novosphingobium pentaromativorans—was capable of growing on pyrene and chrysene as sole source of carbon. A 16S rDNA library was derived from the consortium to identify noncultured bacteria. Among 86 clones screened, 20 were affiliated to different bacterial species–genera. Only three strains were represented in the screened clones. Eighty-five percent of clones and strains were affiliated to Alphaproteobacteria and Betaproteobacteria; among them, several were affiliated to bacterial species known for their PAH degradation activities such as those belonging to the Sphingomonadaceae. Finally, three genes involved in the degradation of aromatic molecules were detected in the consortium and two in IAFILS9. This study provides information on the bacterial composition of a HWM PAH-degrading consortium and its dynamics in a TLP biosystem during PAH degradation.     

Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation during Bioremediation of Heavily Creosote-Contaminated Soil

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.     

Environmental Factors Shape Sediment Anammox Bacterial Communities in Hypernutrified Jiaozhou Bay,China

Bacterial anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. Because ongoing eutrophication of coastal bays contributes significantly to the formation of low-oxygen zones, monitoring of the anammox bacterial community offers a unique opportunity for assessment of anthropogenic perturbations in these environments. The current study used targeting of 16S rRNA and hzo genes to characterize the composition and structure of the anammox bacterial community in the sediments of the eutrophic Jiaozhou Bay, thereby unraveling their diversity, abundance, and distribution. Abundance and distribution of hzo genes revealed a greater taxonomic diversity in Jiaozhou Bay, including several novel clades of anammox bacteria. In contrast, the targeting of 16S rRNA genes verified the presence of only “Candidatus Scalindua,” albeit with a high microdiversity. The genus “Ca. Scalindua” comprised the apparent majority of active sediment anammox bacteria. Multivariate statistical analyses indicated a heterogeneous distribution of the anammox bacterial assemblages in Jiaozhou Bay. Of all environmental parameters investigated, sediment organic C/organic N (OrgC/OrgN), nitrite concentration, and sediment median grain size were found to impact the composition, structure, and distribution of the sediment anammox bacterial community. Analysis of Pearson correlations between environmental factors and abundance of 16S rRNA and hzo genes as determined by fluorescent real-time PCR suggests that the local nitrite concentration is the key regulator of the abundance of anammox bacteria in Jiaozhou Bay sediments.Anaerobic ammonium oxidation (anammox, NH₄⁺ + NO₂⁻ → N₂ + 2H₂O) was proposed as a missing N transformation pathway decades ago. It was found 20 years later to be mediated by bacteria in artificial environments, such as anaerobic wastewater processing systems (see reference 32 and references therein). Anammox in natural environments was found even more recently, mainly in O₂-limited environments such as marine sediments (28, 51, 54, 67, 69) and hypoxic or anoxic waters (10, 25, 39-42). Because anammox may remove as much as 30 to 70% of fixed N from the oceans (3, 9, 64), this process is potentially as important as denitrification for N loss and bioremediation (41, 42, 73). These findings have significantly changed our understanding of the budget of the marine and global N cycles as well as involved pathways and their evolution (24, 32, 35, 72). Studies indicate variable anammox contributions to local or regional N loss (41, 42, 73), probably due to distinct environmental conditions that may influence the composition, abundance, and distribution of the anammox bacteria. However, the interactions of anammox bacteria with their environment are still poorly understood.The chemolithoautotrophic anammox bacteria (64, 66) comprise the new Brocadiaceae family in the Planctomycetales, for which five Candidatus genera have been described (see references 32 and 37 and references therein): “Candidatus Kuenenia,” “Candidatus Brocadia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia.” Due to the difficulty of cultivation and isolation, anammox bacteria are not yet in pure culture. Molecular detection by using DNA probes or PCR primers targeting the anammox bacterial 16S rRNA genes has thus been the main approach for the detection of anammox bacteria and community analyses (58). However, these studies revealed unexpected target sequence diversity and led to the realization that due to biased coverage and specificity of most of the PCR primers (2, 8), the in situ diversity of anammox bacteria was likely missed. Thus, the use of additional marker genes for phylogenetic analysis was suggested in hopes of better capturing the diversity of this environmentally important group of bacteria. By analogy to molecular ecological studies of aerobic ammonia oxidizers, most recent studies have attempted to include anammox bacterium-specific functional genes. All anammox bacteria employ hydrazine oxidoreductase (HZO) (= [Hzo]₃) to oxidize hydrazine to N₂ as the main source for a useable reductant, which enables them to generate proton-motive force for energy production (32, 36, 65). Phylogenetic analyses of Hzo protein sequences revealed three sequence clusters, of which the cladistic structure of cluster 1 is in agreement with the anammox bacterial 16S rRNA gene phylogeny (57). The hzo genes have emerged as an alternative phylogenetic and functional marker for characterization of anammox bacterial communities (43, 44, 57), allowing the 16S rRNA gene-based investigation methods to be corroborated and improved.The contribution of anammox to the removal of fixed N is highly variable in estuarine and coastal sediments (50). For instance, anammox may be an important pathway for the removal of excess N (23) or nearly negligible (48, 54, 67, 68). This difference may be attributable to a difference in the structure and composition of anammox bacterial communities, in particular how the abundance of individual cohorts depends on particular environmental conditions. Anthropogenic disturbance with variable source and intensity of eutrophication and pollution may further complicate the anammox bacterium-environment relationship.Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides (harmful algal blooms), species loss, and contamination with toxic chemicals and harmful microbes (14, 15, 21, 61, 71). Due to different sources of pollution and various levels of eutrophication across Jiaozhou Bay (mariculture, municipal and industrial wastewater, crude oil shipyard, etc.), a wide spectrum of environmental conditions may contribute to a widely varying community structure of anammox bacteria. This study used both 16S rRNA and hzo genes as targets to measure their abundance, diversity, and spatial distribution and assess the response of the resident anammox bacterial community to different environmental conditions. Environmental factors with potential for regulating the sediment anammox microbiota are discussed.     

Illumina Sequencing of 16S rRNA Tag Revealed Spatial Variations of Bacterial Communities in a Mangrove Wetland

The microbial community plays an essential role in the high productivity in mangrove wetlands. A proper understanding of the spatial variations of microbial communities will provide clues about the underline mechanisms that structure microbial groups and the isolation of bacterial strains of interest. In the present study, the diversity and composition of the bacterial community in sediments collected from four locations, namely mudflat, edge, bulk, and rhizosphere, within the Mai Po Ramsar Wetland in Hong Kong, SAR, China were compared using the barcoded Illumina paired-end sequencing technique. Rarefaction results showed that the bulk sediment inside the mature mangrove forest had the highest bacterial α-diversity, while the mudflat sediment without vegetation had the lowest. The comparison of β-diversity using principal component analysis and principal coordinate analysis with UniFrac metrics both showed that the spatial effects on bacterial communities were significant. All sediment samples could be clustered into two major groups, inner (bulk and rhizosphere sediments collected inside the mangrove forest) and outer mangrove sediments (the sediments collected at the mudflat and the edge of the mangrove forest). With the linear discriminate analysis scores larger than 3, four phyla, namely Actinobacteria, Acidobacteria, Nitrospirae, and Verrucomicrobia, were enriched in the nutrient-rich inner mangrove sediments, while abundances of Proteobacteria and Deferribacterias were higher in outer mangrove sediments. The rhizosphere effect of mangrove plants was also significant, which had a lower α-diversity, a higher amount of Nitrospirae, and a lower abundance of Proteobacteria than the bulk sediment nearby.     

Bacterial Degraders of Polycyclic Aromatic Hydrocarbons Isolated from Salt-Contaminated Soils and Bottom Sediments in Salt Mining Areas

Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical- and salt-producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1–1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.     

Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil

Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [¹³C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli, but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two- and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.     

福建红树林湿地鸟类区系研究

红树林湿地是热带、亚热带海岸带的重要湿地类型。由于红树林是自然辅助供能的高生产率的生态系统 ,具有高光合率、高呼吸率和高归还率的三高特点 ,为红树林区底栖动物的生活提供了能量来源 ,丰富的底栖动物又为鸟类等高等动物提供了充足的饵料来源。红树林湿地特殊的环境特点 ,使之成为咸淡水交迭的环境下生存的动植物、微生物丰富的基因库[10 ] 。因此 ,红树林区域在生物多样性研究中具有重要价值。在香港米埔红树林保护区 ,鸟类学家对当地鸟类的迁徙和生态进行了 30多年的研究[15,16 ] ,2 0世纪 80年代后对广东、广西、海南等地主要红树…     

多环芳烃污染生物修复研究进展

多环芳烃(polycyclic aromatic hydrocarbon,PAHs)是一类对环境有严重危害的持久性有机污染物。具有高生物富集性、致癌性、致毒性和难降解性,修复治理PAHs污染环境备受国内外政府及学者的关注。目前主要采用物理、化学以及生物方法对多环芳烃污染的土壤和水体进行修复。其中生物修复是一种高效、经济和生态可承受的环保技术,具有成本低、无二次污染等优点。本文从植物修复、微生物修复以及植物-微生物联合修复方面,阐述了国内外生物修复PAHs污染的最新研究进展。指出了生物修复PAHs污染环境需要进一步解决的问题,并对未来发展趋势进行了展望。     

Effect of Elodea nuttallii Roots on Bacterial Communities and MMHg Proportion in a Hg Polluted Sediment

The objective of this study was to assess the effect of a rooted macrophyte Elodea nuttallii on rhizosphere bacterial communities in Hg contaminated sediments. Specimens of E. nuttallii were exposed to sediments from the Hg contaminated Babeni reservoir (Olt River, Romania) in our microcosm. Plants were allowed to grow for two months until they occupied the entirety of the sediments. Total Hg and MMHg were analysed in sediments where an increased MMHg percentage of the total Hg in pore water of rhizosphere sediments was found. E. nuttallii roots also significantly changed the bacterial community structure in rhizosphere sediments compared to bulk sediments. Deltaproteobacteria dominated the rhizosphere bacterial community where members of Geobacteraceae within the Desulfuromonadales and Desulfobacteraceae were identified. Two bacterial operational taxonomic units (OTUs) which were phylogenetically related to sulfate-reducing bacteria (SRB) became abundant in the rhizosphere. We suggest that these phylotypes could be potentially methylating bacteria and might be responsible for the higher MMHg percentage of the total Hg in rhizosphere sediments. However, SRB were not significantly favoured in rhizosphere sediments as shown by qPCR. Our findings support the hypothesis that rooted macrophytes created a microenvironment favorable for Hg methylation. The presence of E. nuttallii in Hg contaminated sediments should therefore not be overlooked.     

Polycyclic Aromatic Hydrocarbons in Coastal Sediment of Klang Strait,Malaysia: Distribution Pattern,Risk Assessment and Sources

Concentration, source, and ecological risk of polycyclic aromatic hydrocarbons (PAHs) were investigated in 22 stations from surface sediments in the areas of anthropogenic pollution in the Klang Strait (Malaysia). The total PAH level in the Klang Strait sediment was 994.02±918.1 µg/kg dw. The highest concentration was observed in stations near the coastline and mouth of the Klang River. These locations were dominated by high molecular weight PAHs. The results showed both pyrogenic and petrogenic sources are main sources of PAHs. Further analyses indicated that PAHs primarily originated from pyrogenic sources (coal combustion and vehicular emissions), with significant contribution from petroleum inputs. Regarding ecological risk estimation, only station 13 was moderately polluted, the rest of the stations suffered rare or slight adverse biological effects with PAH exposure in surface sediment, suggesting that PAHs are not considered as contaminants of concern in the Klang Strait.     

Structural and Functional Variation in Genetic Systems for Catabolism of Polycyclic Aromatic Hydrocarbons in Pseudomonas putida Strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

Detection of a Reproducible,Single-Member Shift in Soil Bacterial Communities Exposed to Low Levels of Hydrogen

Soil is exposed to hydrogen when symbiotic rhizobia in legume root nodules cannot recycle the hydrogen that is generated during nitrogen fixation. The hydrogen emitted is most likely taken up by free-living soil bacteria that use hydrogen as an energy source, though the bacteria that do this in situ remain unclear. In this study, we investigated the effect of hydrogen exposure on the bacteria of two different soils in a microcosm setup designed to simulate hydrogen-emitting root nodules. Although the size and overall composition of the soil bacterial community did not significantly alter after hydrogen exposure, one ribotype increased in relative abundance within each soil. This single-ribotype shift was identified by generating multiple terminal restriction fragment length polymorphism (T-RFLP) profiles of 16S rRNA genes from each soil sample, with gene sequence confirmation to identify terminal restriction fragments. The increased abundance of a single ribotype after hydrogen exposure, within an otherwise similar community, was found in replicate samples taken from each microcosm and was reproducible across replicate experiments. Similarly, only one member of the soil bacterial community increased in abundance in response to hydrogen exposure in soil surrounding the root nodules of field-grown soybean (Glycine max). The ribotypes that increased after hydrogen exposure in each soil system tested were all from known hydrogen-oxidizing lineages within the order Actinomycetales. We suggest that soil actinomycetes are important utilizers of hydrogen at relevant concentrations in soil and could be key contributors to soil''s function as a sink in the global hydrogen cycle.Soil is the major sink in the global hydrogen cycle and accounts for approximately 75 to 80% of uptake from the atmosphere (7, 11). Soil is such a strong sink that the atmospheric mixing ratio of molecular hydrogen, H₂, is hemispherically asymmetric because of the greater landmass in the Northern Hemisphere (11). Many nitrogen-fixing bacteria that form symbiotic relationships with legume plants cannot recycle the H₂ that is generated during N₂ fixation (2, 13). Most of the H₂ emitted from legume root nodules is taken up by the surrounding soil, within a few centimeters of the nodule surface, and is not released to the atmosphere (20). Although the H₂ emitted by the rhizobial symbionts costs the legume approximately 5% of its daily photosynthate and “represents a significant investment by the plant” (9), there is growing evidence to suggest that soil exposed to H₂ is beneficial to plant growth, separate from the benefits derived from N₂ fixation (8, 10, 28). Previously, La Favre and Focht have hypothesized that “the hydrogen which is evolved during N₂ fixation represents an additional energy input into the plant-soil ecosystem… since metabolism of H₂ by chemolithotrophic bacteria results in an input of fixed carbon to the system” (20). A number of studies have found that when H₂ is taken up by soil, net CO₂ fixation occurs at the rate of 7 to 8 nmol CO₂ per g of soil per h (22, 34). For a legume fixing 200 kg of atmospheric N₂ per hectare, over 200,000 liters of H₂ could be released into the legume''s rhizosphere over the duration of the growing season and CO₂ fixation could result in an extra 25 kg of soil carbon fixed per hectare (9, 10, 28).Many bacteria isolated from soil are able to utilize H₂ as an energy source (2, 5-7, 21), and free-living bacteria are most likely responsible for the H₂ uptake observed by soil surrounding legume roots (22). Adding a bacterial energy source, such as H₂, could affect the microbial population size, as has been observed previously (34), but more specific shifts within the bacterial community may occur if just the microorganisms able to utilize the energy source multiply. Their activity could also have downstream consequences specifically on other members of the community. Most H₂-oxidizing cultures have required enrichment with concentrations of H₂ that are not environmentally relevant and therefore cannot be assumed to be carrying out H₂ oxidation at much lower, naturally occurring concentrations (5-7). Recent surveys of microbes present in soil samples, via their nucleic acids, have revealed many novel bacterial inhabitants that have been little studied and thus may also be contributing to the repertoire of bacterial soil processes, such as H₂ uptake (16). A recent study into the effect of H₂ on soil bacteria focused on a few groups of H₂-oxidizing, autotrophic bacteria and thus ignored many other H₂ utilizers potentially present in soil (34).There are now many ways of characterizing the entire microbial community in environmental samples, either via their entire genomic content, though metagenomic analysis of soil is difficult at present, or via analysis of the lineages present according to 16S rRNA gene sequences, or ribotypes (36). A recent study comparing high-throughput pyrosequencing of 16S rRNA genes and an easily accessible profiling method, known as terminal restriction fragment length polymorphism (T-RFLP), found the simpler profiles were appropriate for comparing the dominant ribotypes in multiple samples (24). Although T-RFLP profiles only provide a simplified snapshot of the dominant members in microbial communities, compared to the deeper analyses provided by microarrays or high-throughput sequencing technologies, T-RFLP profiling is a cost-effective, reproducible, and robust method of “fingerprinting” many soil samples rapidly and efficiently (14, 24, 25, 32).In this study, we chose to assess the dominant members of the soil bacterial community via T-RFLP profiles of ribotypes present in H₂-treated and control soils to avoid a narrow focus on well-studied H₂ oxidizers. We investigated the bacterial community structure in two different soils, utilizing a microcosm setup with concentrations of H₂ calculated to occur in the rhizosphere of N₂-fixing legumes, to determine whether common responses to H₂ exposure could be predicted from soils that differ by climate, edaphic characteristics, and starting communities. Soil in microcosms has previously been shown to have similar H₂ uptake properties to soil close to H₂-emitting legume nodules (9), but we also complemented our plant-free microcosm work with an examination of soil collected from the root systems of field-grown soybean (Glycine max (L.) Merr.).