Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

Succession of Phenotypic, Genotypic, and Metabolic Community Characteristics during In Vitro Bioslurry Treatment of Polycyclic Aromatic Hydrocarbon-Contaminated Sediments

Dredged harbor sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) was removed from the Milwaukee Confined Disposal Facility and examined for in situ biodegradative capacity. Molecular techniques were used to determine the successional characteristics of the indigenous microbiota during a 4-month bioslurry evaluation. Ester-linked phospholipid fatty acids (PLFA), multiplex PCR of targeted genes, and radiorespirometry techniques were used to define in situ microbial phenotypic, genotypic, and metabolic responses, respectively. Soxhlet extractions revealed a loss in total PAH concentrations of 52%. Individual PAHs showed reductions as great as 75% (i.e., acenapthene and fluorene). Rates of ¹⁴C-PAH mineralization (percent/day) were greatest for phenanthrene, followed by pyrene and then chrysene. There was no mineralization capacity for benzo[a]pyrene. Ester-linked phospholipid fatty acid analysis revealed a threefold increase in total microbial biomass and a dynamic microbial community composition that showed a strong correlation with observed changes in the PAH chemistry (canonical r² of 0.999). Nucleic acid analyses showed copies of genes encoding PAH-degrading enzymes (extradiol dioxygenases, hydroxylases, and meta-cleavage enzymes) to increase by as much as 4 orders of magnitude. Shifts in gene copy numbers showed strong correlations with shifts in specific subsets of the extant microbial community. Specifically, declines in the concentrations of three-ring PAH moieties (i.e., phenanthrene) correlated with PLFA indicative of certain gram-negative bacteria (i.e., Rhodococcus spp. and/or actinomycetes) and genes encoding for naphthalene-, biphenyl-, and catechol-2,3-dioxygenase degradative enzymes. The results of this study suggest that the intrinsic biodegradative potential of an environmental site can be derived from the polyphasic characterization of the in situ microbial community.     

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 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.     

Polycyclic Aromatic Hydrocarbon (PAH) Contamination in the Urban Topsoils of Shenyang,China

Polycyclic aromatic hydrocarbons (PAHs) in the surface urban soils of Shenyang in Northeastern China were investigated. The total concentration of the PAHs ranged from 0.09 to 8.35 mg kg^?1, with an average value of 1.51 ± 1.64 mg kg^?1. 3–5-ring PAHs accounted for 90% of total PAHs. The functional areas, such as the industrial regions (4.95 mg kg^?1) and main roads (1.56 mg kg^?1), as well as the administrative divisions, including the districts of Shenhe (1.49 mg kg^?1), Heping (2.08 mg kg^?1), and Tiexi (2.14 mg kg^?1), were heavily polluted by PAHs. The diagnostic ratios and principal component analysis (PCA) for PAHs indicate that the pollutants probably originated primarily from coal combustion and petroleum sources. The Nemerow composite index, used to assess environmental quality, shows that the soil samples were heavily polluted with PAHs, and although 52.8% of the soil sampling sites were safe, 47.2% of the soil sampling sites registered different grades of PAH pollution. The PAH contamination in Shenyang emphasizes the need for controlling fossil fuel combustion and traffic exhaust.     

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     

Association of Microbial Community Composition and Activity with Lead, Chromium, and Hydrocarbon Contamination

Microbial community composition and activity were characterized in soil contaminated with lead (Pb), chromium (Cr), and hydrocarbons. Contaminant levels were very heterogeneous and ranged from 50 to 16,700 mg of total petroleum hydrocarbons (TPH) kg of soil⁻¹, 3 to 3,300 mg of total Cr kg of soil⁻¹, and 1 to 17,100 mg of Pb kg of soil⁻¹. Microbial community compositions were estimated from the patterns of phospholipid fatty acids (PLFA); these were considerably different among the 14 soil samples. Statistical analyses suggested that the variation in PLFA was more correlated with soil hydrocarbons than with the levels of Cr and Pb. The metal sensitivity of the microbial community was determined by extracting bacteria from soil and measuring [³H]leucine incorporation as a function of metal concentration. Six soil samples collected in the spring of 1999 had IC₅₀ values (the heavy metal concentrations giving 50% reduction of microbial activity) of approximately 2.5 mM for CrO₄²⁻ and 0.01 mM for Pb²⁺. Much higher levels of Pb were required to inhibit [¹⁴C]glucose mineralization directly in soils. In microcosm experiments with these samples, microbial biomass and the ratio of microbial biomass to soil organic C were not correlated with the concentrations of hydrocarbons and heavy metals. However, microbial C respiration in samples with a higher level of hydrocarbons differed from the other soils no matter whether complex organic C (alfalfa) was added or not. The ratios of microbial C respiration to microbial biomass differed significantly among the soil samples (P < 0.05) and were relatively high in soils contaminated with hydrocarbons or heavy metals. Our results suggest that the soil microbial community was predominantly affected by hydrocarbons.     

Degradation of Polycyclic Aromatic Hydrocarbons in the Rhizosphere of Festuca arundinacea and Associated Microbial Community Changes

A phytoremediation growth chamber study was conducted to evaluate the contribution of soil microbial diversity to the contaminant degradation. Target contaminant removal from soil was assessed by monitoring concentrations of polycyclic aromatic hydrocarbons (PAHs), along with changes in the bacterial community structure over a time period of 10 months in the presence of tall fescue (Festuca arundinacea). Enhanced degradation of PAHs was observed in rhizosphere soil, with a maximum reduction in pyrene at a rate 36% higher than that noted for the unvegetated control. The dissipation of < 4-ring PAHs, 4-ring PAHs, and > 4-ring PAHs in unvegetated soil was 70%, 54%, and 49% respectively, whereas a higher dissipation rate was observed in tall fescue treated soil of 78%, 68%, and 61% at the end of the study. Microbial enumeration results showed greater total bacterial numbers and PAH-degrading bacteria in rhizosphere soil when compared to unvegetated soil. The results from the terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that there was a shift in the rhizosphere bacterial community during the phytoremediation process.     

Catabolic Gene Probe Analysis of an Aquifer Microbial Community Degrading Creosote-Related Polycyclic Aromatic and Heterocyclic Compounds

Abstract The biodegradation of a mixture of several creosote-related compounds, p-cresol, phenanthrene, fluorene, and carbazole was examined in columns containing aquifer sands. The aquifer material, itself, had an effect on the migration of the test compounds, with p-cresol being retarded the least, followed by carbazole, then fluorene, and finally phenanthrene. The biodegradation of all the compounds was greatly enhanced by the inclusion of p-cresol (10 ppm) in the substrate mixture. Associated with this enhanced degradation was a 100-fold increase in the total culturable bacterial population, and increases in the xylE- and ndoB-positive bacterial populations of more than three orders of magnitude. The products of these two genes are involved in the degradation of monocyclic and polycyclic aromatic compounds, respectively. In columns that did not receive p-cresol, there was no significant change in either the total culturable bacterial population density or the xylE-positive bacterial population, but there were significant increases of one to two orders of magnitude in the ndoB-positive bacterial populations. The results suggest that the ndoB gene probe can detect bacteria capable of utilizing phenanthrene, carbazole, and possibly fluorene. Received: 26 January 1996; Accepted: 20 June 1996     

Impact of Irradiation and Polycyclic Aromatic Hydrocarbon Spiking on Microbial Populations in Marine Sediment for Future Aging and Biodegradability Studies

Experiments were carried out to develop methods to generate well-characterized, polycyclic aromatic hydrocarbon (PAH)-spiked, aged but minimally altered sediments for fate, biodegradation, and bioavailability experiments. Changes in indigenous bacterial populations were monitored in mesocosms constructed of relatively clean San Diego Bay sediments, with and without exposure to gamma radiation, and then spiked with five different PAHs and hexadecane. While phenanthrene and chrysene degraders were present in the unspiked sediments and increased during handling, PAH spiking of nonirradiated sediments led to dramatic increases in their numbers. Phenotypic characterization of isolates able to grow on phenanthrene or chrysene placed them in several genera of marine bacteria: Vibrio, Marinobacter or Cycloclasticus, Pseudoalteromonas, Marinomonas, and Halomonas. This is the first time that marine PAH degraders have been identified as the latter two genera, expanding the diversity of marine bacteria with this ability. Even at the highest irradiation dose (10 megarads), heterotrophs and endospore formers reappeared within weeks. However, while bacteria from the unirradiated sediments had the capacity to both grow on and mineralize ¹⁴C-labeled phenanthrene and chrysene, irradiation prevented the reappearance of PAH degraders for up to 4 months, allowing spikes to age onto the sediments, which can be used to model biodegradation in marine sediments.     

Microbial Transformation of Polycyclic Aromatic Hydrocarbons in Pristine and Petroleum-Contaminated Sediments

To determine rates of microbial transformation of polycyclic aromatic hydrocarbons (PAH) in freshwater sediments, ¹⁴C-labeled PAH were incubated with samples from both pristine and petroleum-contaminated streams. Evolved ¹⁴CO₂ was trapped in KOH, unaltered PAH and polar metabolic intermediate fractions were quantitated after sediment extraction and column chromatography, and bound cellular ¹⁴C was measured in sediment residues. Large fractions of ¹⁴C were incorporated into microbial cellular material; therefore, measurement of rates of ¹⁴CO₂ evolution alone would seriously underestimate transformation rates of [¹⁴C]naphthalene and [¹⁴C]anthracene. PAH compound turnover times in petroleum-contaminated sediment increased from 7.1 h for naphthalene to 400 h for anthracene, 10,000 h for benz(a)anthracene, and more than 30,000 h for benz(a)pyrene. Turnover times in uncontaminated stream sediment were 10 to 400 times greater than in contaminated samples, while absolute rates of PAH transformation (micrograms of PAH per gram of sediment per hour) were 3,000 to 125,000 times greater in contaminated sediment. The data indicate that four- and five-ring PAH compounds, several of which are carcinogenic, may persist even in sediments that have received chronic PAH inputs and that support microbial populations capable of transforming two- and three-ring PAH compounds.     

Integrated Approach for Polycyclic Aromatic Hydrocarbon Solubilization from the Soil Matrix to Enhance Bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are known to be toxic to living organisms and have been identified as carcinogenic. In this study, a pathway of surfactant flushing, chemical oxidation, and biological treatment is proposed to remediate the soils polluted with the hydrophobic PAHs. Different surfactants such as Tween 80, Brij 35, sodium dodecyl sulfate (SDS), and polyethylene glycol (PEG) 6000 were tested in order to increase the PAH solubilization from the soil matrix. The maximum desorption efficiency of naphthalene and anthracene were found to be 56.5% and 59%, respectively, when Brij and SDS were used. The soluble PAH in the aqueous phase was amended with sodium thiosulfate (3%) to oxidize the PAH into a more bioavailable form. The chemical oxidation with subsequent biodegradation by Pseudomonas aeruginosa exhibited the relatively high PAH degradation rate (1.24 times higher) when compared with chemical oxidation alone. These results display the efficiency of chemical pretreatment of PAH-contaminated soil for improved bioremediation.     

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

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

Soil Type-Dependent Responses to Phenanthrene as Revealed by Determining the Diversity and Abundance of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes by Using a Novel PCR Detection System

A novel PCR primer system that targets a wide range of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD_α) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHD_α gene amplicons from soil DNA. Cloning and sequencing showed the dominance of phnAc genes in the contaminated Luvisol. In contrast, high diversity of PAH-RHD_α genes of Gram-positive and Gram-negative bacteria was observed in the phenanthrene-spiked Cambisol. Quantitative real-time PCR based on the same primers revealed that 63 days after phenanthrene spiking, PAH-RHD_α genes were 1 order of magnitude more abundant in the Luvisol than in the Cambisol, while they were not detected in both control soils. In conclusion, sequence analysis of the amplicons obtained confirmed the specificity of the novel primer system and revealed a soil type-dependent response of PAH-RHD_α gene-carrying soil bacteria to phenanthrene spiking.Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic compounds composed of two or more fused aromatic rings. Although PAHs are ubiquitous in the environment (from natural oil seeps, brush fires, and plant derivatives), anthropogenic activities, such as disposal of coal-processing waste, mining accidents, petroleum wastes, and vehicle exhaust, have drastically increased their occurrence in the environment. The fate of PAHs in soil is of great interest due to their potential for bioaccumulation, persistence, transport, and toxicity. Microbe-driven aerobic degradation of PAHs is well documented (15-17). The diversity of PAH-degrading genes in soils is assumed to be huge, but the extent of diversity and how it is influenced by different soil types or their history and type of pollution are not yet fully explored. Knowledge of the genes coding for dioxygenase enzymes that catalyze the primary step of PAH degradation by incorporating molecular oxygen into the aromatic nucleus is an essential prerequisite to unraveling the contributions of microbial population networks to transformation, assimilation, and degradation of organic chemicals in soil. Recently, the complete genomes of several PAH-degrading bacteria became available and allowed new insights into degradative pathways (6, 18, 36). Organic pollutants also serve as nutrients for those microbes that have the appropriate genetic makeup to utilize them, resulting in their increased metabolic activity and abundance (4, 14). In the last decade, impressive progress was seen in techniques that allow cultivation-independent analysis of microbial communities and thus overcome the most severe limitations in studying microbial communities in natural habitats, namely, that only a rather small portion of microbes are accessible to standard cultivation conditions (1, 29). For more than a decade, cultivation-independent approaches have also been employed to unravel the responses of microbial communities in soils and sediments to PAH pollution. In all these studies, PCR amplification of PAH-degrading gene fragments from nucleic acids directly extracted from environmental samples was used to explore the abundance and diversity of PAH ring-hydroxylating dioxygenase (PAH-RHD_α) genes (4, 8, 9, 13, 14, 22, 34, 37). Despite the known biases of PCR amplification from mixed templates, these techniques allow highly sensitive and specific detection even from minute amounts of nucleic acids. In order to select suitable primer systems, previously published primer systems were analyzed for their ranges of target sequences. The existing primer systems were found to have limitations, as they often target only a rather narrow range of sequences, e.g., nahAc- or phnAc-type sequences (21, 34) or only PAH-RHD_α genes from Gram-negative bacteria (3, 13). In other studies, two-primer systems were used to target PAH-RHD_α genes of both Gram-positive and Gram-negative bacteria (4, 37). Only one primer system targeting the Rieske gene fragment was described that amplified a small fragment from PAH-RHD_α genes from both Gram-negative and Gram-positive bacteria (24). However, the amplicon size was only 78 bp and the primer might also target genes coding for dioxygenases that attack nonpolar aromatic compounds, such as benzene, toluene, and xylene. Therefore, this work aimed to design an improved primer system that targets PAH-RHD_α genes from both Gram-positive and Gram-negative bacteria and provides larger amplicon sizes. The novel primer system was tested in silico and validated by sequencing cloned PAH-RHD_α genes amplified from total-community (TC) DNA and was used in endpoint and quantitative real-time PCR (qPCR) formats. The primer system was also applied to study the responses of soil microbial communities in two different soils (a Cambisol and a Luvisol representing typical arable soils in Central Europe with different texture compositions) to artificial phenanthrene pollution.     

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.     

微生物降解多环芳烃的研究进展

多环芳烃(PAHs)是具有严重危害的环境污染物质。介绍PAHs的降解菌,降解机理和PAHs的生物修复方面的研究进展。土壤中PAHs的生物修复被认为是解决污染的有效方法,目前,菲的生物降解途径已经比较清楚,但对结构更为复杂的多环芳烃研究较少。文章还对消除环境中多环芳烃的相关生物技术提出展望。     

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.     

Microbial Community Responses to Atmospheric Carbon Dioxide Enrichment in a Warm-Temperate Forest

Forest productivity depends on nutrient supply, and sustained increases in forest productivity under elevated carbon dioxide (CO₂) may ultimately depend on the response of microbial communities to changes in the quantity and chemistry of plant-derived substrates, We investigated microbial responses to elevated CO₂ in a warm-temperate forest under free-air CO₂ enrichment for 5 years (1997–2001). The experiment was conducted on three 30 m diameter plots under ambient CO₂ and three plots under elevated CO₂ (200 ppm above ambient). To understand how microbial processes changed under elevated CO₂, we assayed the activity of nine extracellular enzymes responsible for the decomposition of labile and recalcitrant carbon (C) substrates and the release of nitrogen (N) and phosphorus (P) from soil organic matter. Enzyme activities were measured three times per year in a surface organic horizon and in the top 15 cm of mineral soil. Initially, we found significant increases in the decomposition of labile C substrates in the mineral soil horizon under elevated CO₂; this overall pattern was present but much weaker in the O horizon. Beginning in the 4th year of this study, enzyme activities in the O horizon declined under elevated CO₂, whereas they continued to be stimulated in the mineral soil horizon. By year 5, the degradation of recalcitrant C substrates in mineral soils was significantly higher under elevated CO₂. Although there was little direct effect of elevated CO₂ on the activity of N- and P-releasing enzymes, the activity of nutrient-releasing enzymes relative to those responsible for C metabolism suggest that nutrient limitation is increasingly regulating microbial activity in the O horizon. Our results show that the metabolism of microbial communities is significantly altered by the response of primary producers to elevated CO₂. We hypothesize that ecosystem responses to elevated CO₂ are shifting from primary production to decomposition as a result of increasing nutrient limitation.     

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.