Enumeration and phylogenetic analysis of polycyclic aromatic hydrocarbon-degrading marine bacteria from Puget sound sediments.

Naphthalene- and phenanthrene-degrading bacteria in Puget Sound sediments were enumerated by most-probable-number enumeration procedures. Sediments from a creosote-contaminated Environmental Protection Agency Superfund Site (Eagle Harbor) contained from 10(4) to 10(7) polycyclic aromatic hydrocarbon (PAH)-degrading bacteria g (dry weight) of sediment-1, whereas the concentration at an uncontaminated site ranged from 10(3) to 10(4) g of sediment(-1). Isolates of PAH-degrading bacteria were obtained from these most-probable-number tubes as well as from sediment samples from noncontaminated sites and from bioreactors enriched with PAHs. The 18 resulting strains were grouped by whole-cell fatty acid analysis into two subgroups. The larger group of strains belonged to the newly described genus Cycloclasticus, whereas the other group contained members of the genus Vibrio. The Cycloclasticus group seems to be widespread in noncontaminated sediments. PAH degradation was confirmed in selected strains on the basis of removal of phenanthrene from growing cultures.     

Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria from Bizerte lagoon sediments, Tunisia

Aims: To characterize polycyclic aromatic hydrocarbon (PAH)‐degrading bacteria from sediments of the Bizerte lagoon, and to determine their ability to resist other pollutants such as antibiotics and heavy metals. Methods and Results: More than 100 strains were isolated for their ability to use fluoranthene as the sole carbon and energy source. Most of them showed antibiotic and heavy metal resistance; 20 representative strains were selected for further analysis. 16S rRNA coding sequences analysis showed that the majority of the selected bacteria (75%) were affiliated to the Gammaproteobacteria. The selected strains also utilized high molecular weight PAHs containing up to four benzene rings and showed different profiles of PAH substrate usage suggesting different PAH degradation pathways. These results are consistent with the fact that nah‐like genes and idoA‐like genes, involved in PAH degradation, were detected in 6 and 1 strains respectively. Conclusions: The Bizerte lagoon, polluted by many human activities, leads to the co‐selection of strains able to cope with multiple contaminants. Significance and Impact of the Study: Polluted areas are often characterized by the concomitant presence of organic pollutants, heavy metals and antibiotics. This study is one of the first showing bacterial strains adapted to multiple contaminants, a promising potential for the development of bioremediation processes.     

Genetic diversity of dioxygenase genes in polycyclic aromatic hydrocarbon-degrading bacteria isolated from mangrove sediments

To investigate the diversity of dioxygenase genes involved in polycyclic aromatic hydrocarbon (PAH)-degradation, a total of 32 bacterial strains were isolated from surface mangrove sediments, from the genera Mycobacterium, Sphingomonas, Terrabacter, Sphingopyxis, Sphingobium and Rhodococcus. Two sets of PCR primers were constructed to detect the nidA-like and nahAc-like sequences of the alpha subunit of the PAH ring-hydroxylating dioxygenase. PCR amplified the DNA fragments from all Gram-positive bacteria by using nidA-like primers and from all Gram-negative bacteria, except two, by using nahAc-like primers. The nidA-like primers showed three subtypes of nidA-like gene: (i) fadA1, clustering with nidA3 from M. vanbaalenii PYR-1, (ii) nidA, clustering with nidA from PYR-1, and (iii) fadA2 clustering with dioxygenase from Arthrobacter sp. FB24. The amplicons detected by nahAc-like primers had high sequence homologies to phnA1a from Sphingomonas sp. CHY-1 and were amplifiable from 8 of the 16 Gram-negative isolates. The primer also generated amplicons that had a 32-36% similarity to phnA1a and 53-93% identity to p-cumate dioxygenase. These results suggest that the nidA-like and nahAc-like genes are prevalent in the PAH-degrading bacteria and that they are useful for determining the presence of PAH-dioxygenase genes in environmental samples.     

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas , Halomonas , Marinobacter , Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus , Alteromonas , Thalassospira , Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously.     

Enrichment and identification of polycyclic aromatic compound-degrading bacteria enriched from sediment samples

The degradation of polycyclic aromatic compounds (PACs) has been widely studied. Knowledge of the degradation of PACs by microbial populations can be utilized in the remediation of contaminated sites. To isolate and identify PAC-degrading bacteria for potential use in future bioremediation programmes, we established a series of PAC enrichments under the same experimental conditions from a single sediment sample taken from a highly polluted estuarine site. Enrichment cultures were established using the pollutants: anthracene, phenanthrene and dibenzothiophene as a sole carbon source. The shift in microbial community structure on each of these carbon sources was monitored by analysis of a time series of samples from each culture using 16S rRNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Significantly, our findings demonstrate that shifts in the constituent species within each degradative community are directly attributable to enrichment with different PACs. Subsequently, we characterized the microorganisms comprising the degradative communities within each enrichment using 16S rRNA sequence data. Our findings demonstrate that the ability to degrade PACs is present in five divisions of the Proteobacteria and Actinobacteria. By determining the precise identity of the PAC-degrading bacterial species isolated from a single sediment sample, and by comparing our findings with previously published research, we demonstrate how bacteria with similar PAC degrading capabilities and 16S rRNA signatures are found in similarly polluted environments in geographically very distant locations, e.g., China, Italy, Japan and Hawaii. Such a finding suggests that geographical barriers do not limit the distribution of key PAC-degrading bacteria; this finding is in accordance with the Baas-Becking hypothesis “everything is everywhere; the environment selects” and may have significant consequences for the global distribution of PAC-degrading bacteria and their use in bioremediation.     

Enrichment versus biofilm culture: a functional and phylogenetic comparison of polycyclic aromatic hydrocarbon-degrading microbial communities

The effect that culture methods have on the diversity of degradative microbial communities is not well understood. We compared conventional batch enrichment with a biofilm culture method for the isolation of polycyclic aromatic hydrocarbon (PAH)-degrading microbial communities from a PAH-contaminated soil. The two methods were assessed by comparing: (i) the diversity of culturable bacteria; (ii) the diversity of PAH-catabolic genes in isolated bacteria; (iii) the inter- and intraspecific diversity of active PAH-catabolic gene classes; (iv) the diversity of bacteria present in 16S rRNA gene libraries generated from RNA extracted from the two communities and soil; and (v) the estimated diversity of active bacteria in the soil and culture systems. Single-strand conformation polymorphism analysis showed that the biofilm culture yielded 36 bacterial and two fungal species compared with 12 bacterial species from the enrichment culture. Application of accumulation and non-parametric estimators to clone libraries generated from 16S rRNA confirmed that the biofilm community contained greater diversity. Sequencing of clones showed that only species from the Proteobacteria were active in the enrichment culture, and that these species were expressing an identical nahAc-like naphthalene dioxygenase. 16S rRNA clones generated from the biofilm community indicated that species from the Cytophaga/Flavobacterium, high G+C bacteria and Proteobacteria were active at the time of sampling, expressing cndA-, nahAc- and phnAc-like naphthalene dioxygenases. The diversity of active species in the biofilm culture system closely matched that in the PAH-contaminated source soil. The results of this study showed that biofilm culture methods are more appropriate for the study of community-level interactions in PAH-degrading microbial communities. The study also indicated that cultivation of microbial communities on solid media might be the primary source of bias in the recovery of diverse species.     

Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soils

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria isolated from PAH-contaminated soils were analyzed genotypically and phenotypically for their capacity for metabolism of naphthalene and other PAH substrates. The methods used for the analyses were DNA hybridization using NAH7-derived gene probes, PAH spray plate assays, ¹⁴C-PAH mineralization assays, and dioxygenase activity assays. The results of the analyses showed a dominant number of PAH-degrading bacteria with a NAH7-like genotype. The results support the continued use of the nahA probe for contaminated soils to monitor the genetic potential of indigenous microorganisms to degrade PAHs. However, the finding of non-it nahA-hybridizing PAH-degrading bacteria show the limitation of NAH7-derived gene probes. Fifteen percent (13/89) of PAH-degrading bacteria isolated were not detected with the nahA gene probe. Four isolates (designated A5PH1, A8AN3, B1PH2, and B10AN1) did not hybridize with any of the NAH7-derived gene probes ( nahA, nahG, nahH, and nahR) used in this study. Considering the numerous unculturable microorganisms in nature and their potential genotypes, NAH7-derived gene probes may underestimate the microbial potential to catabolize PAHs. This necessitates development of new gene probes for enumeration and isolation of PAH-degrading bacteria to better understand the in situ microbial potential to degrade PAHs.     

Products from the incomplete metabolism of pyrene by polycyclic aromatic hydrocarbon-degrading bacteria

Pyrene is a regulated pollutant at sites contaminated with polycyclic aromatic hydrocarbons (PAH). It is mineralized by some bacteria but is also transformed to nonmineral products by a variety of other PAH-degrading bacteria. We examined the formation of such products by four bacterial strains and identified and further characterized the most apparently significant of these metabolites. Pseudomonas stutzeri strain P16 and Bacillus cereus strain P21 transformed pyrene primarily to cis-4,5-dihydro-4,5-dihydroxypyrene (PYRdHD), the first intermediate in the known pathway for aerobic bacterial mineralization of pyrene. Sphingomonas yanoikuyae strain R1 transformed pyrene to PYRdHD and pyrene-4,5-dione (PYRQ). Both strain R1 and Pseudomonas saccharophila strain P15 transform PYRdHD to PYRQ nearly stoichiometrically, suggesting that PYRQ is formed by oxidation of PYRdHD to 4,5-dihydroxypyrene and subsequent autoxidation of this metabolite. A pyrene-mineralizing organism, Mycobacterium strain PYR-1, also transforms PYRdHD to PYRQ at high initial concentrations of PYRdHD. However, strain PYR-1 is able to use both PYRdHD and PYRQ as growth substrates. PYRdHD strongly inhibited phenanthrene degradation by strains P15 and R1 but had only a minor effect on strains P16 and P21. At their aqueous saturation concentrations, both PYRdHD and PYRQ severely inhibited benzo[a]pyrene mineralization by strains P15 and R1. Collectively, these findings suggest that products derived from pyrene transformation have the potential to accumulate in PAH-contaminated systems and that such products can significantly influence the removal of other PAH. However, these products may be susceptible to subsequent degradation by organisms able to metabolize pyrene more extensively if such organisms are present in the system.     

Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites

Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.     

两株多环芳烃降解菌协同对菲-镉污染的去除特性北大核心

【目的】从污染土壤中分离筛选一株多环芳烃降解菌,并探究其与Pseudomonas aeruginosa B6-2构建的混菌体系对菲-镉复合污染的修复效能,以及微生物代谢特性对不同镉浓度赋存的响应特性,以期为复合污染的生物修复提供优良菌株资源及应用技术参考。【方法】采用富集驯化、筛选纯化方法得到一株多环芳烃降解菌,通过生理生化特征和16S rRNA基因序列分析进行鉴定。利用高效液相色谱法和电感耦合等离子体质谱法评估不同镉浓度赋存下各反应体系对菲和镉的去除效能;通过菌体细胞形态的扫描电镜观测及菌株代谢活性检测,探讨镉胁迫对菲生物降解过程的影响机制。【结果】筛选得到一株具有重金属耐受性和多环芳烃高效降解菌SZ-3,经鉴定为节杆菌属;降解菌协同体系(M)具有良好的菲降解效能和抗镉胁迫优势。镉胁迫浓度为0.5、10 mg/L时,M对菲和镉的去除率分别高于85%、80%;镉胁迫浓度为25、50 mg/L时,2种污染物的去除率均大于65%。扫描电镜分析表明,镉胁迫导致菌体表面粗糙且出现不同程度变形,菌体间黏附性和聚集性提高。反应周期内,邻苯二酚1,2-双加氧酶活性与电子传递体系活性随镉浓度增加而降低,两者变化与菲降解速率变化一致。【结论】Arthrobacter sp.SZ-3是一株PAHs高效降解菌,能与Pseudomonas aeruginosa B6-2协同高效修复菲-镉复合污染,随着初始镉胁迫浓度增加,混菌协同对目标污染物去除的优势显著。     

Isolation and characterization of heavy polycyclic aromatic hydrocarbon-degrading bacteria adapted to electrokinetic conditions

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria capable of growing under electrokinetic conditions were isolated using an adjusted acclimation and enrichment procedure based on soil contaminated with heavy PAHs in the presence of an electric field. Their ability to degrade heavy PAHs under an electric field was individually investigated in artificially contaminated soils. The results showed that strains PB4 (Pseudomonas fluorescens) and FB6 (Kocuria sp.) were the most efficient heavy PAH degraders under electrokinetic conditions. They were re-inoculated into a polluted soil from an industrial site with a PAH concentration of 184.95 mg kg^?1. Compared to the experiments without an electric field, the degradation capability of Pseudomonas fluorescens and Kocuria sp. was enhanced in the industrially polluted soil under electrokinetic conditions. The degradation extents of total PAHs were increased by 15.4 and 14.0 % in the electrokinetic PB4 and FB6 experiments (PB4 + EK and FB6 + EK) relative to the PB4 and FB6 experiments without electrokinetic conditions (PB4 and FB6), respectively. These results indicated that P. fluorescens and Kocuria sp. could efficiently degrade heavy PAHs under electrokinetic conditions and have the potential to be used for the electro-bioremediation of PAH-contaminated soil, especially if the soil is contaminated with heavy PAHs.     

Molecular characterization of dioxygenases from polycyclic aromatic hydrocarbon-degrading Mycobacterium spp

Polycyclic aromatic hydrocarbon (PAH)-degrading genes nidA and nidB that encode the alpha and beta subunits of the aromatic ring-hydroxylating dioxygenase have been cloned and sequenced from Mycobacterium vanbaalenii PYR-1 [Khan et al., Appl. Environ Microbiol. 67 (2001) 3577-3585]. In this study, the presence of nidA and nidB in 12 other Mycobacterium or Rhodococcus strains was investigated. Initially, all strains were screened for their ability to degrade PAHs by a spray plate method, and for the presence of the dioxygenase Rieske center region by polymerase chain reaction (PCR). Only Mycobacterium sp. PAH 2.135 (RJGII-135), M. flavescens PYR-GCK (ATCC 700033), M. gilvum BB1 (DSM 9487) and M. frederiksbergense FAn9T (DSM 44346), all previously known PAH degraders, were positive in both tests. From the three positive strains, complete open reading frames of the nidA and nidB genes were amplified by PCR, using primers designed according to the known nidA and nidB sequences from PYR-1, cloned in the pBAD/Thio-TOPO vector and sequenced. The sequences showed >98% identity with the M. vanbaalenii PYR-1 nidA and nidB genes. Southern DNA-DNA hybridization using nidA and nidB probes from PYR-1 revealed that there is more than one copy of nidA and nidB genes in the strains PYR-1, BB1, PYR-GCK and FAn9T. However, only one copy of each gene was observed in PAH2.135.     

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 10³ to 10⁵ bacteria ml^–1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.Abbreviations BTEX benzene, toluene, ethylbenzene, andp-xylene - HPLC high performance liquid chromatography - GAC granular activated carbon     

Phylogenetic comparison of two polycyclic aromatic hydrocarbon-degrading mycobacteria.

Two mycobacterial strains previously isolated from fossil-fuel-contaminated environments and shown to degrade four- and/or five-ring polycyclic aromatic hydrocarbons were further characterized. The two strains, PYR-I and RJGII-135, had similar growth characteristics, colony morphologies, and scotochromogenic pigmentations. DNA amplification fingerprints obtained with total genomic DNA indicated some strain similarities but with several distinctly different bands. Moreover, phylogenetic analysis based upon essentially full-length 16S rRNA gene sequences separates the two strains as distinct species within the fast-growing group of mycobacteria. Although both strains are thermosensitive, strain PYR-I has the bulged U between positions 184 and 193 characteristic of thermotolerant mycobacteria. Both strains are of potential use for reintroduction into and bioremediation of polycyclic aromatic hydrocarbon-contaminated soils.     

Pseudomonas, the dominant polycyclic aromatic hydrocarbon-degrading bacteria isolated from Antarctic soils and the role of large plasmids in horizontal gene transfer

Twenty-two polycyclic aromatic hydrocarbon (PAH)-degrading bacterial strains were isolated from Antarctic soils with naphthalene or phenanthrene as a sole carbon source, while no degrader was obtained from an unpolluted sampling site. Phylogenetic analysis showed that all belonged to the genus Pseudomonas except one that was identified as the genus of Rahnella. Some of them were closely related to previously reported cold-tolerant species, while some were separated in deeply rooted branches and represent new strains. All these strains showed a high efficiency to degrade naphthalene at 4 degrees C, and some additionally degraded phenanthrene. Using degenerate primers and polymerase chain reaction (PCR) amplification, ndo gene encoding naphthalene dioxygenase (NDO) was detected from all the isolates. Phylogenetic analysis grouped these genes into two clusters which shared 94% similarity to each other, and showed about 97% similarity within a cluster. However, no obvious difference was observed with mesophilic ndo genes; this indicates that the host cell is pivotal in cold adaptation. In addition, the mismatch between 16S rRNA and NDO phylogenetic trees strongly indicates horizontal gene transfer among these isolates and may have happened in situ. Further, Southern hybridization and plasmid curing confirmed that ndo genes were located on a large self-transmissible plasmid, which can be transferred to a mesophilic strains. The transconjugants acquired the ability to utilize naphthalene and phenanthrene. Results of this article imply that Pseudomonas plays an important role in PAH biodegradation in Antarctic soils, and the related genes might be originally transferred from outside Antarctica and spread among indigenous species.     

Isolation and characterization of polycyclic aromatic hydrocarbon-degrading bacteria associated with the rhizosphere of salt marsh plants

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon and energy. Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were characterized using a variety of phenotypic, morphologic, and molecular properties. Identification of the isolates based on their fatty acid profiles and partial 16S rRNA gene sequences assigned them to three main bacterial groups: gram-negative pseudomonads; gram-positive, non-spore-forming nocardioforms; and the gram-positive, spore-forming group, Paenibacillus. Genomic digest patterns of all isolates were used to determine unique isolates, and representatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation from Pseudomonas putida NCIB 9816-4, Comamonas testosteroni GZ42, Sphingomonas yanoikuyae B1, and Mycobacterium sp. strain PY01. None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform isolates showed homology to the PY01 genes, and only members of the pseudomonad group showed homology to the NCIB 9816-4 or GZ42 probes. The Paenibacillus isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the three groups showed that the phenanthrene-enriched isolates are able to utilize a greater number of PAHs than are the naphthalene-enriched isolates. Inoculating two of the gram-positive isolates to a marine sediment slurry spiked with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to the two- and three-ringed PAHs and biphenyl. This study indicates that the rhizosphere of salt marsh plants contains a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremediation of contaminated sediments.     

A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.     

Selection of a plant-bacterium pair as a novel tool for rhizostimulation of polycyclic aromatic hydrocarbon-degrading bacteria

We developed a novel procedure for the selection of a microbe-plant pair for the stable and efficient degradation of naphthalene. Based on the rationale that root exudate is the best nutrient source available in soil, the grass (Lolium multiflorum) cultivar Barmultra was selected because of its abilities to produce a highly branched root system, root deeply, and carry a high population of Pseudomonas spp. bacteria on its roots. Starting with a mixture of total rhizobacteria from grass-like vegetation collected from a heavily polluted site and selecting for stable naphthalene degradation as well as for efficient root colonization, Pseudomonas putida strain PCL1444 was isolated. The strain's ability to degrade naphthalene was shown to be stable in the rhizosphere. Moreover, it had superior root-colonizing properties because, after the inoculation of grass seedlings, it appeared to colonize the root tip up to 100-fold better than the efficient root colonizer Pseudomonas fluorescens WCS365. Strain PCL1444 uses root exudate as the dominant nutrient source because the presence of grass seedlings in soil results in up to a 10-fold increase of PCL1444 cells. Moreover, the root colonized by strain PCL1444 was able to penetrate through an agar layer, resulting in the degradation of naphthalene underneath this layer. In addition, the inoculation of grass seeds or seedlings with PCL1444 protected them against naphthalene phytotoxicity. Finally, this plant-microbe combination appeared able to degrade naphthalene from soil that was heavily polluted with a complex mixture of polycyclic aromatic hydrocarbons. To our knowledge, this is the first time that a naturally occurring bacterium has been selected for the combination of the abilities to degrade a pollutant and colonize plant roots. We suggest that the principle described here, to select a bacterium which combines efficient root colonization with a beneficial activity, also can be used to improve the selection of other more efficient plant-bacterium pairs for beneficial purposes such as biocontrol, biofertilization, and phytostimulation.     

Diversity among aromatic hydrocarbon-degrading bacteria and their meta-cleavagegenes

Sixty-one strains of bacteria capable of growth on 4-methyl benzoic acid (29 isolates) ornaphthalene (32 isolates) as the sole source of carbon and energy were isolated from sedimentsand water samples from the River Tyne, UK. Random amplification of polymorphic DNA fromgenomic DNA extracted from the different strains demonstrated that 14 of the 4-methylbenzoate-degrading isolates were unique and the remainder fell into seven groups containing twoor three isolates that produced identical banding patterns. Thirteen of the naphthalene-degradingisolates were unique and nine groups with two or three identical representatives encompassed allother isolates. Screening of the bacterial strains for the presence of genes homologous to xylE , nahC and bphC by polymerase chain reaction and dot blot hybridizationdemonstrated that most strains harboured xylE - and/or nahC -like genes and only asingle isolate was found that did not harbour any of these genes. None of the isolates harboured bphC -like genes. It was concluded that, while considerable diversity existed in host strainsisolated using a single simple enrichment procedure, the extradiol dioxygenase genes involved inaromatic ring cleavage, present in these strains, were conserved to a considerable degree.