Diffuse PAH contamination of surface soils: environmental occurrence,bioavailability, and microbial degradation

The purpose of this review is to recognize the scientific and environmental importance of diffuse pollution with polycyclic aromatic hydrocarbons (PAHs). Diffuse PAH pollution of surface soil is characterized by large area extents, low PAH concentrations, and the lack of point sources. Urban and pristine topsoils receive a continuous input of pyrogenic PAHs, which induces a microbial potential for PAH degradation. The significance of this potential in relation to black carbon particles, PAH bioaccessibility, microbial PAH degradation, and the fate of diffuse PAHs in soil is discussed. Finally, the state-of-the-art methods for future investigations of the microbial degradation of diffuse PAH pollution are reviewed.     

Illumina-based analysis of microbial community diversity

Microbes commonly exist in milieus of varying complexity and diversity. Although cultivation-based techniques have been unable to accurately capture the true diversity within microbial communities, these deficiencies have been overcome by applying molecular approaches that target the universally conserved 16S ribosomal RNA gene. The recent application of 454 pyrosequencing to simultaneously sequence thousands of 16S rDNA sequences (pyrotags) has revolutionized the characterization of complex microbial communities. To date, studies based on 454 pyrotags have dominated the field, but sequencing platforms that generate many more sequence reads at much lower costs have been developed. Here, we use the Illumina sequencing platform to design a strategy for 16S amplicon analysis (iTags), and assess its generality, practicality and potential complications. We fabricated and sequenced paired-end libraries of amplified hyper-variable 16S rDNA fragments from sets of samples that varied in their contents, ranging from a single bacterium to highly complex communities. We adopted an approach that allowed us to evaluate several potential sources of errors, including sequencing artifacts, amplification biases, non-corresponding paired-end reads and mistakes in taxonomic classification. By considering each source of error, we delineate ways to make biologically relevant and robust conclusions from the millions of sequencing reads that can be readily generated by this technology.     

Molecular characterization of anaerobic microbial communities from benzene-degrading sediments under methanogenic conditions

Anaerobic benzene degradation was confirmed in microbial communities enriched from Baltimore Harbor (Baltimore, MD) sediments under methanogenic conditions. Molecular characterization based on 16S rDNA gene sequences revealed that the strains in the communities were diversely affiliated with such phylogenetic branches as the Bacteroidetes, Euryarchaeota, Firmicutes, and Thermotogae phyla. Of interest was that the majority of the microbial populations detected in these cultures were closely related to the members of dechlorinating microbial communities. Further, some of those species were previously found in naphthalene- or phenanthrene-degrading methanogenic communities. Finally, this result could be used to design targeted isolation strategies for anaerobic benzene-degrading strains under methanogenic conditions.     

Polycyclic Aromatic Hydrocarbon (PAH) Degradation Coupled to Methanogenesis

Baltimore Harbor (Baltimore, MD) sediments were utilized to initiate anaerobic enrichment cultures with polycyclic aromatic hydrocarbons (PAHs) in the absence of supplementary electron acceptors. Cultures amended with naphthalene and phenanthrene exhibited sustained, transferable degradation of the PAHs. Bromoethanesulfonic acid, a selective inhibitor of methanogenesis, inhibited the degradation of 200 μm naphthalene and phenanthrene; molecular characterization based on 16S rRNA sequences confirmed that methanogenic Archaea were eliminated, thus providing evidence that methanogenesis is involved in the degradation pathway. Revisions requested 16 November 2005; Revisions received 14 December 2005     

Impact of increasing NaCl concentrations on the performance and community composition of two anaerobic reactors

The anaerobic treatment of saline effluents using halophilic and halotolerant microbial consortia is of major interest. Inhibition of anaerobic digestion is known to occur at high salt content. However, it seems that the suitable adaptation of an anaerobic sludge makes possible the treatment of saline wastewater. In this study, a non-saline anaerobic sludge was inoculated in two anaerobic batch reactors operating with a different substrate (distillery vinasse and ethanol) and subjected to increasing NaCl concentrations. The performance of the digesters appeared to be highly dependent on the nature of the substrate, and a similar level of inhibition (i.e. around 90% of the specific loading rate and specific methanogenic activity) was stated at 10 g l⁻¹ of NaCl with distillery vinasse and 60 g l⁻¹ of NaCl with ethanol. The characterization of the microflora and its adaptation to increasing NaCl conditions were also investigated using molecular tools based on the analysis of genomic 16S rDNA. The microbial communities revealed a high diversity that could be maintained in both reactors despite the increase in NaCl concentrations.     

Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis

The microbial community structure of pig manure slurry (PMS) was determined with comparative analysis of 202 bacterial, 44 archaeal and 33 eukaryotic small subunit (SSU) rDNA partial sequences. Based on a criterion of 97% of sequence similarity, the phylogenetic analyses revealed a total of 108, eight and five phylotypes for the Bacteria, Archaea and Eukarya lineages, respectively. Only 36% of the bacterial phylotypes were closely related (>or=97% similarity) to any previously known sequence in databases. The bacterial groups most often represented in terms of phylotype and clone abundance were the Eubacterium (22% of total sequences), the Clostridium (15% of sequences), the Bacillus-Lactobacillus-Streptococcus subdivision (20% of sequences), theMycoplasma and relatives (10% of sequences) and the Flexibacter-Cytophaga-Bacteroides (20% of sequences). The global microbial community structure and phylotype diversity show a close relationship to the pig gastrointestinal tract ecosystem whereas phylotypes from the Acholeplasma-Anaeroplasma and the Clostridium purinolyticum groups appear to be better represented in manure. Archaeal diversity was dominated by three phylotypes clustering with a group of uncultured microorganisms of unknown activity and only distantly related to the Thermoplasmales and relatives. Other Archaea were methanogenic H2/CO2 utilisers. No known acetoclastic Archaea methanogen was found. Eukaryotic diversity was represented by a pluricellular nematode, two Alveolata, a Blastocystis and an Entamoebidae. Manure slurry physico-chemical characteristics were analysed. Possible inhibitory effects of acetate, sulphide and ammonia concentrations on the microbial anaerobic ecosystem are discussed.     

Isolation of phenanthrene-degrading bacteria and characterization of phenanthrene metabolites

Three aerobic bacterial consortia GY2, GS3 and GM2 were enriched from polycyclic aromatic hydrocarbon-contaminated soils with water-silicone oil biphasic systems. An aerobic bacterial strain utilizing phenanthrene as the sole carbon and energy source was isolated from bacterial consortium GY2 and identified as Sphingomonas sp. strain GY2B. Within 48 h and at 30°C the strain metabolized 99.1% of phenanthrene (100 mg/l) added to batch culture in mineral salts medium and the cell number increased by about 40-fold. Three metabolites 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, were identified by gas chromatographic mass spectrometry and UV–visible spectroscopy analysis. A degradation pathway was proposed based on the identified metabolites. In addition to phenanthrene, strain GY2B could use other aromatic compounds such as naphthalene, 2-naphthol, salicylic acid, catechol, phenol, benzene and toluene as a sole source of carbon and energy.     

石油污染土壤水改旱田后污染物组份及微生物群落结构变化

对沈抚灌区水改旱田不同年限土壤的石油污染物浓度及组分进行了分析,并采用变性梯度凝胶电泳和磷脂脂肪酸分析方法,分析了污染土壤微生物群落结构的变化.结果表明：1)石油污染土壤水改旱田后,年限越长,总多环芳烃在总石油烃中所占的比重越大,高分子量多环芳烃在总多环芳烃中所占比重也越大;2)总磷脂脂肪酸量与总石油烃呈显著正相关,与总多环芳烃相关性不显著;3)两种方法对土壤微生物群落结构的分析得出的结论一致,石油污染土壤微生物群落结构主要与其相对地理位置有关,当污染物的浓度达到一定程度时,土壤微生物群落结构会发生明显的改变.     

Molecular characterization of sulfate-reducing bacteria in anaerobic hydrocarbon-degrading consortia and pure cultures using the dissimilatory sulfite reductase (dsrAB) genes

The characterization of sulfate-reducing bacteria (SRBs) is presented using the dissimilatory sulfite reductase (dsrAB) gene from various samples capable of mineralizing petroleum components. These samples include several novel, sulfidogenic pure cultures which degrade alkanes, toluene, and tribromophenol. Additionally, we have sulfidogenic consortia which re-mineralize benzene, naphthalene, 2-methylnaphthalene, and phenanthrene as a sole carbon source. In this study, 22 new dsrAB genes were cloned and sequenced. The dsrAB genes from our pollutant-degrading cultures or consortia were distributed among known SRBs and previously described dsrAB environmental clones, suggesting that many biodegradative SRBs are phylogenetically distinct and geographically wide spread. Specifically, the same dsrAB gene was discovered in independently established consortia capable of benzene, phenanthrene, and methylnaphthalene degradation, indicating that this particular SRB may be a key player in anaerobic degradation of hydrocarbons in the environment.     

Dynamic changes in nahAc gene copy numbers during degradation of naphthalene in PAH-contaminated soils

Many bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) contain the nahAc gene that encodes a component of multimeric naphthalene dioxygenases. Because the nahAc gene is highly conserved, this gene serves as a potential biomarker for PAH degradation activity. The aim of this research was to examine the relationship between the rate of naphthalene degradation and the copy number of the nahAc gene in soils using conventional and real-time PCR. Four sets of degenerate primers for real-time PCR were designed based on the nahAc DNA sequences of 33 bacterial species. Before addition of naphthalene, copy numbers of the nahAc gene were below the detection limits of the assay at 5×10³ copy numbers per gram of soil, but increased by over a thousand fold to 10⁷ copies after 6 days of exposure to naphthalene vapors (approximately 30 ppm soil water concentration). Two unreported naphthalene dioxygenase homologs were found in the naphthalene-spiked soil by cloning and sequencing of the PCR products from the nahAc primers. Results of these experiments demonstrate the highly dynamic changes that occur in soil microbial communities after exposure to naphthalene and suggest that there is a direct relationship between gene copy numbers and degradation rates for naphthalene in PAH-contaminated soils.     

Self-induced bio-potential and graphite electron accepting conditions enhances petroleum sludge degradation in bio-electrochemical system with simultaneous power generation

Bio-electrochemical treatment (BET) documented effective degradation of real field petroleum sludge over the conventional anaerobic treatment (AnT). BET (41.08%) operation showed enhanced total petroleum hydrocarbons (TPH) removal over AnT (20.72%). Aromatic fraction visualized higher removal (75.54%) compared to other TPH fractions viz., aliphatics, asphaltenes and NSO (nitrogen, sulfur and oxygen) during BET operation. Higher ring aromatics (5-6) documented easy degradation in BET, while AnT was limited to lower ring (2-3) compounds. Voltammetric analysis evidenced simultaneous redox behavior during BET operation due to presence of graphite electrode as electron acceptor, while AnT showed extended reduction behavior only. Self-induced primary and secondary oxidation reactions and capacitive-deionization might have enhanced the degradation capability of BET. BET documented higher charge/capacitance (2810 mJ/1120 mF) than AnT (450 mJ/180 mF). Power output corroborated well with observed results supporting BET performance as fuel cell. Electrodes offer a potential alternative electron acceptor for promoting the degradation of organic contaminants.     

土壤中高环多环芳烃微生物降解的研究进展

微生物修复是去除土壤中多环芳烃(PAHs)的主要措施。本文以微生物修复PAHs污染土壤的理论基础及其难点为主线,全面综述了土壤中高环PAHs的微生物降解机理。近年来,富集分离得到的以高环PAHs为唯一碳源和能源的优势降解菌逐渐增多,其中,主要是代谢降解四环PAHs的单株降解菌,一些降解菌还能以共代谢方式利用五环PAHs。高环PAHs污染土壤修复的一个难点是其低生物可利用性,微生物通过释放生物表面活性剂、形成生物膜以及分泌胞外多糖提高高环PAHs的生物可利用性,从而加速其降解。真菌和细菌联合作用能增强污染土壤实地修复的效果。因此,通过微生物修复技术来去除土壤中PAHs具有环境友好性、经济适用性以及可持续应用性。     

Performance comparison of genetic markers for high‐throughput sequencing‐based biodiversity assessment in complex communities

Metabarcode surveys of DNA extracted from environmental samples are increasingly popular for biodiversity assessment in natural communities. Such surveys rely heavily on robust genetic markers. Therefore, analysis of PCR efficiency and subsequent biodiversity estimation for different types of genetic markers and their corresponding primers is important. Here, we test the PCR efficiency and biodiversity recovery potential of three commonly used genetic markers – nuclear small subunit ribosomal DNA (18S), mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (mt16S) – using 454 pyrosequencing of a zooplankton community collected from Hamilton Harbour, Ontario. We found that biodiversity detection power and PCR efficiency varied widely among these markers. All tested primers for COI failed to provide high‐quality PCR products for pyrosequencing, but newly designed primers for 18S and 16S passed all tests. Furthermore, multiple analyses based on large‐scale pyrosequencing (i.e. 1/2 PicoTiter plate for each marker) showed that primers for 18S recover more (38 orders) groups than 16S (10 orders) across all taxa, and four vs. two orders and nine vs. six families for Crustacea. Our results showed that 18S, using newly designed primers, is an efficient and powerful tool for profiling biodiversity in largely unexplored communities, especially when amplification difficulties exist for mitochondrial markers such as COI. Universal primers for higher resolution markers such as COI are still needed to address the possible low resolution of 18S for species‐level identification.     

4S polycyclic aromatic hydrocarbon receptor (glycine N‐methyltransferase) and the aryl hydrocarbon receptor nuclear translocator (hypoxia inducible factor‐1β) interaction in Chinese hamster ovary and rat hepatoma cells: 4S PAH‐R/ARNT hetero‐oligomers?

Rat CYP1A1 promoter‐luciferase, transiently transfected wild‐type and 4S PAH receptor (glycine N‐methyl transferase, GNMT)‐transformed Chinese hamster ovary (CHO) cells were exposed to benzo[a]pyrene and assayed for luciferase activity as an indicator of CYP1A1 promoter activity. CHO cells transformed with the rat 4S PAH receptor/GNMT expression vector had twice the induction level of luciferase activity with respect to wild‐type CHO cells in concert with previously published reports that the 4S PAH receptor/GNMT mediates benzo[a]pyrene induction of CYP1A1 gene expression. Lysates of GNMT‐transformed CHO cells and wild‐type H4IIE rat hepatoma cells exposed to benzo[a]pyrene were immuno‐precipitated with anti‐GNMT antibodies, separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membrane for Western blot analysis with anti‐aryl hydrocarbon receptor nuclear translocator (ARNT, HIF‐1β) antibodies. Results of this analysis indicated that the 4S PAH receptor/GNMT forms a hetero‐oligomer (dimer?) with ARNT/HIF‐1β which dissociates in the presence of B[a]P. These observations further indicate the role of GNMT (which has been shown to be multifunctional) and B[a]P in the induction of CYP1A1 and also a potential role of GNMT in the modulation of hypoxia inducible factor‐1 function with respect to the HIF‐1β subunit (ARNT). J. Cell. Biochem. 112: 2015–2018, 2011. © 2011 Wiley‐Liss, Inc.     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Molecular Detection of Novel Anammox Bacterial Clusters in the Sediments of the Shallow Freshwater Lake Taihu

Previous studies have demonstrated the wide occurrence of anaerobic ammonium oxidizing (anammox) bacteria; however, there is very limited information on the distribution of these bacteria in freshwater habitats. In this study, the anammox bacterial communities were detected by molecular analysis targeting the 16S rRNA genes in the sediments of Lake Taihu, a large and shallow eutrophic freshwater lake in China. The recovery of specific 16S rRNA sequences with two stable monophyletic clusters indicated that anammox bacteria were present in Lake Taihu. A phylogenetic analysis indicated that these two groups represent two novel lineages within the first subgroup of anammox bacteria, independent of the treeing methods. High intra-lake variability in anammox bacterial diversity and community composition was observed, in particular, based on a 1% cut-off of 16S rRNA sequence variation. The spatial variability was largely related to the substrate availability, which was denoted by the correlations between the relative abundance of the two Taihu anammox bacterial groups and the concentrations of ammonium and nitrite. This indicates that the niche differentiation of anammox bacteria is linked to the environmental heterogeneity. These findings suggest that the freshwater lakes may accommodate different anammox bacterial communities and, thus, expand our knowledge on the diversity and distribution of anammox bacteria. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.