Orbital-resolved observation of singlet fission.

Singlet fission1-13 may boost photovoltaic efficiency14-16 by transforming a singlet exciton into two triplet excitons and thereby doubling the number of excited charge carriers. The primary step of singlet fission is the ultrafast creation of the correlated triplet pair17. Whereas several mechanisms have been proposed to explain this step, none has emerged as a consensus. The challenge lies in tracking the transient excitonic states. Here we use time- and angle-resolved photoemission spectroscopy to observe the primary step of singlet fission in crystalline pentacene. Our results indicate a charge-transfer mediated mechanism with a hybridization of Frenkel and charge-transfer states in the lowest bright singlet exciton. We gained intimate knowledge about the localization and the orbital character of the exciton wave functions recorded in momentum maps. This allowed us to directly compare the localization of singlet and bitriplet excitons and decompose energetically overlapping states on the basis of their orbital character. Orbital- and localization-resolved many-body dynamics promise deep insights into the mechanics governing molecular systems18-20 and topological materials21-23.

Ultrafast Momentum-Resolved Hot Electron Dynamics in the Two-Dimensional Topological Insulator Bismuthene.

Two-dimensional quantum spin Hall (QSH) insulators are a promising material class for spintronic applications based on topologically protected spin currents in their edges. Yet, they have not lived up to their technological potential, as experimental realizations are scarce and limited to cryogenic temperatures. These constraints have also severely restricted characterization of their dynamical properties. Here, we report on the electron dynamics of the novel room-temperature QSH candidate bismuthene after photoexcitation using time- and angle-resolved photoemission spectroscopy. We map the transiently occupied conduction band and track the full relaxation pathway of hot photocarriers. Intriguingly, we observe photocarrier lifetimes much shorter than those in conventional semiconductors. This is ascribed to the presence of topological in-gap states already established by local probes. Indeed, we find spectral signatures consistent with these earlier findings. Demonstration of the large band gap and the view into photoelectron dynamics mark a critical step toward optical control of QSH functionalities.

Yeast diversity during the fermentation of Andean chicha: A comparison of high-throughput sequencing and culture-dependent approaches.

Diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Five hundred and ninety six yeasts were isolated by classical microbiological methods and 16 species identified by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing (HTS) of D1/D2 26S rRNA gene amplicons from chicha samples detected more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical analysis of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species dominated the pre-fermentative steps and abounded in fermented samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with very few reads in the pyrosequencing analysis.

Future for probiotic science in functional food and dietary supplement development.

PURPOSE OF REVIEW: The purpose of this study is to provide an update of probiotic science evolving from classical approaches to the development of next-generation probiotics, parallel to advances in the understanding of the complexity of the gut microbiome and its role in human health. RECENT FINDINGS: The probiotic concept is based on the notion that the gut ecosystem contributes to human physiology and, consequently, its modulation may help to maintain health and reduce disease risk. The understanding of the complexity of the gut microbiota and the specific components associated with progression from health to disease is rapidly increasing, thanks to the use of high-throughput and next-generation sequencing techniques in progressively better controlled epidemiological studies. Evidence on microbiome-mediated effects by intervention with classical probiotics on humans is, however, limited. The new information is helping to set a rationale for selection of a next generation of probiotics. Candidates include Clostridia clusters IV, XIVa and XVIII, Faecalibacterium prausnitzii, Akkermansia muciniphila and Bacteroides uniformis, the effects of which have been evaluated in preclinical trials with promising results for inflammatory and diet-related disorders. Yet, the extent to which new probiotic formulations consisting of nonconventional indigenous gut bacteria will be effective on humans at a population level or in personalized nutrition strategies remains to be explored. SUMMARY: Understanding the role that indigenous intestinal bacteria and their ecological interactions play in human health and disease based on epidemiological, intervention and mechanistic studies will provide a robust rationale for selection of probiotic strains and facilitate the optimization of integrated dietary strategies to efficiently modulate the human gut microbiome, leading to improvements in nutrition and clinical practice.

Observation of ultrafast interfacial Meitner-Auger energy transfer in a Van der Waals heterostructure.

Atomically thin layered van der Waals heterostructures feature exotic and emergent optoelectronic properties. With growing interest in these novel quantum materials, the microscopic understanding of fundamental interfacial coupling mechanisms is of capital importance. Here, using multidimensional photoemission spectroscopy, we provide a layer- and momentum-resolved view on ultrafast interlayer electron and energy transfer in a monolayer-WSe2/graphene heterostructure. Depending on the nature of the optically prepared state, we find the different dominating transfer mechanisms: while electron injection from graphene to WSe2 is observed after photoexcitation of quasi-free hot carriers in the graphene layer, we establish an interfacial Meitner-Auger energy transfer process following the excitation of excitons in WSe2. By analysing the time-energy-momentum distributions of excited-state carriers with a rate-equation model, we distinguish these two types of interfacial dynamics and identify the ultrafast conversion of excitons in WSe2 to valence band transitions in graphene. Microscopic calculations find interfacial dipole-monopole coupling underlying the Meitner-Auger energy transfer to dominate over conventional Förster- and Dexter-type interactions, in agreement with the experimental observations. The energy transfer mechanism revealed here might enable new hot-carrier-based device concepts with van der Waals heterostructures.

Evolutionary convergence and nitrogen metabolism in Blattabacterium strain Bge, primary endosymbiont of the cockroach Blattella germanica.

Bacterial endosymbionts of insects play a central role in upgrading the diet of their hosts. In certain cases, such as aphids and tsetse flies, endosymbionts complement the metabolic capacity of hosts living on nutrient-deficient diets, while the bacteria harbored by omnivorous carpenter ants are involved in nitrogen recycling. In this study, we describe the genome sequence and inferred metabolism of Blattabacterium strain Bge, the primary Flavobacteria endosymbiont of the omnivorous German cockroach Blattella germanica. Through comparative genomics with other insect endosymbionts and free-living Flavobacteria we reveal that Blattabacterium strain Bge shares the same distribution of functional gene categories only with Blochmannia strains, the primary Gamma-Proteobacteria endosymbiont of carpenter ants. This is a remarkable example of evolutionary convergence during the symbiotic process, involving very distant phylogenetic bacterial taxa within hosts feeding on similar diets. Despite this similarity, different nitrogen economy strategies have emerged in each case. Both bacterial endosymbionts code for urease but display different metabolic functions: Blochmannia strains produce ammonia from dietary urea and then use it as a source of nitrogen, whereas Blattabacterium strain Bge codes for the complete urea cycle that, in combination with urease, produces ammonia as an end product. Not only does the cockroach endosymbiont play an essential role in nutrient supply to the host, but also in the catabolic use of amino acids and nitrogen excretion, as strongly suggested by the stoichiometric analysis of the inferred metabolic network. Here, we explain the metabolic reasons underlying the enigmatic return of cockroaches to the ancestral ammonotelic state.

Microbiota and volatilome of dry-cured pork loins manufactured with paprika and reduced concentration of nitrite and nitrate.

Dry-cured pork loin is a very popular meat product in Mediterranean countries. Pork-loin is manufactured rubbing curing salts, nitrite and nitrate, and spices on the surface of the loin which is then dry-cured or smoked for several months. Although nitrite-derived compounds are crucial for the microbiological safety and development of a distinct flavour, there have been recent concerns about the adverse health effects of nitrite-derived compounds driving to the reduction of curing agents in meat products. In this study, we have evaluated the differences in microbiota and aroma of dry-cured pork loins manufactured with or without paprika and reduced ingoing amounts of nitrate and nitrite. Staphylococcus dominated the microbiota of pork loins without paprika, regardless of the nitrite and nitrate reduction. On the contrary, the reduction of nitrite and nitrate in loins with paprika had an important effect on the microbiota. In these loins a codominance of Staphylococcus and Bacillus together by Enterobacteriaceae occurred. Moreover, paprika addition and reduction of nitrite and nitrate seemed to promote proliferation of lactic acid bacteria. Occurrence of these genera was correlated with the generation of free amino acids and their derived volatile compounds setting clear differences in the aroma profile of dry-cured loins.

Blattabacteria, the endosymbionts of cockroaches, have small genome sizes and high genome copy numbers.

Blattabacteria are intracellular endosymbionts of cockroaches and primitive termites that belong to the class Flavobacteria and live only in specialized cells in the abdominal fat body of their hosts. In the present study we determined genome sizes as well as genome copy numbers for the endosymbionts of three cockroach species, Blattella germanica, Periplaneta americana and Blatta orientalis. The sole presence of blattabacteria in the fat body was demonstrated by rRNA-targeting techniques. The genome sizes of the three blattabacteria were determined by pulsed field gel electrophoresis. The resulting total genome sizes for the three symbionts were all approximately 650 +/- 15 kb. Comparison of the genome sizes with those of free-living Bacteroidetes shows extended reduction, as occurs in other obligatory insect endosymbionts. Genome copy numbers were determined based on cell counts and determination of DNA amounts via quantitative PCR. Values between 10.2 and 18.3 and between 323 and 353 were found for the symbionts of P. americana and B. orientalis respectively. Polyploidy in intracellular bacteria may play a significant role in the genome reduction process.

The modular network structure of the mutational landscape of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is associated with the sequential accumulation of acquired genetic alterations. Although at diagnosis cytogenetic alterations are frequent in AML, roughly 50% of patients present an apparently normal karyotype (NK), leading to a highly heterogeneous prognosis. Due to this significant heterogeneity, it has been suggested that different molecular mechanisms may trigger the disease with diverse prognostic implications. We performed whole-exome sequencing (WES) of tumor-normal matched samples of de novo AML-NK patients lacking mutations in NPM1, CEBPA or FLT3-ITD to identify new gene mutations with potential prognostic and therapeutic relevance to patients with AML. Novel candidate-genes, together with others previously described, were targeted resequenced in an independent cohort of 100 de novo AML patients classified in the cytogenetic intermediate-risk (IR) category. A mean of 4.89 mutations per sample were detected in 73 genes, 35 of which were mutated in more than one patient. After a network enrichment analysis, we defined a single in silico model and established a set of seed-genes that may trigger leukemogenesis in patients with normal karyotype. The high heterogeneity of gene mutations observed in AML patients suggested that a specific alteration could not be as essential as the interaction of deregulated pathways.

Bifidobacterium pseudocatenulatum CECT 7765 Reduces Obesity-Associated Inflammation by Restoring the Lymphocyte-Macrophage Balance and Gut Microbiota Structure in High-Fat Diet-Fed Mice.

BACKGROUND/OBJECTIVES: The role of intestinal dysbiosis in obesity-associated systemic inflammation via the cross-talk with peripheral tissues is under debate. Our objective was to decipher the mechanisms by which intervention in the gut ecosystem with a specific Bifidobacterium strain reduces systemic inflammation and improves metabolic dysfunction in obese high-fat diet (HFD) fed mice. METHODS: Adult male wild-type C57BL-6 mice were fed either a standard or HFD, supplemented with placebo or Bifidobacterium pseudocatenulatum CECT 7765, for 14 weeks. Lymphocytes, macrophages and cytokine/chemokine concentrations were quantified in blood, gut, liver and adipose tissue using bead-based multiplex assays. Biochemical parameters in serum were determined by ELISA and enzymatic assays. Histology was assessed by hematoxylin-eosin staining. Microbiota was analyzed by 16S rRNA gene pyrosequencing and quantitative PCR. RESULTS: B. pseudocatenulatum CECT 7765 reduced obesity-associated systemic inflammation by restoring the balance between regulatory T cells (Tregs) and B lymphocytes and reducing pro-inflammatory cytokines of adaptive (IL-17A) and innate (TNF-α) immunity and endotoxemia. In the gut, the bifidobacterial administration partially restored the HFD-induced alterations in microbiota, reducing abundances of Firmicutes and of LPS-producing Proteobacteria, paralleled to reductions in B cells, macrophages, and cytokines (IL-6, MCP-1, TNF-α, IL-17A), which could contribute to systemic effects. In adipose tissue, bifidobacterial administration reduced B cells whereas in liver the treatment increased Tregs and shifted different cytokines (MCP-1 plus ILP-10 in adipose tissue and INF-γ plus IL-1ß in liver). In both tissues, the bifidobacteria reduced pro-inflammatory macrophages and, TNF-α and IL-17A concentrations. These effects were accompanied by reductions in body weight gain and in serum cholesterol, triglyceride, glucose and insulin levels and improved oral glucose tolerance and insulin sensitivity in obese mice. CONCLUSIONS: Here, we provide evidence of the immune cellular mechanisms by which the inflammatory cascade associated with diet-induced obesity is attenuated by the administration of a specific Bifidobacterium strain and that these effects are associated with modulation of gut microbiota structure.

Fluorescence based rRNA sensor systems for detection of whole cells of Saccharomonospora spp. and Thermoactinomyces spp.

Airborne thermophilic actinomycetes (TPAs) are a growing hygienic challenge in different occupational situations e.g. large scale composting. This study describes first results of a new approach for highly specific and rapid detection of organisms of this group using fluorescently labelled oligonucleotide probes as sensors for whole cells. Three genus-specific 16S rRNA-targeted probes, two for Saccharomonospora spp. and one for Thermoactinomyces spp. were developed and evaluated in a fluorescence in situ hybridisation (FISH) format with agar-grown whole cells. For optimal sensitivity and specificity of FISH, conditions for cell wall permeabilisation and hybridisation stringency were evaluated independently for both genera. Performing specified pretreatment protocols, all three probes yielded strong fluorescence signals. However, the relative fraction of detectable cells or spores clearly depended on the single bacterial species. The probes can serve as cell sensors for direct detection of TPAs in natural samples.

Novosphingobium hassiacum sp. nov., a new species isolated from an aerated sewage pond.

The taxonomy of two strains W-51T and W-52 isolated from a wastewater treatment plant was investigated in a polyphasic approach. The yellow pigmented gram-negative organism contained a quinone system with mainly ubiquinone Q-10, and the polar lipid profile contained a sphingoglycolipid suggesting that both strains belonged to the the alpha-4 subclass of the Proteobacteria. The polar lipid profile consisted furthermore of phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylcholine and of minor amounts of phosphatidylglycerol and phosphatidylmonomethylethanolamine. Sequencing of the 16S rRNA gene supported the allocation into the genus Novosphingobium, together with the type strains of N. subterraneum, N. aromaticivorans, N. stygium, and N. capsulatum, showing similarities of 97.3%, 97.0%, 95.7% and 96.2%, respectively. This allocation was supported by the polyamine profile, which consisted mainly of spermidine. The analysis of the fatty acids revealed 2-OH 13:0, 2-OH 14:0 and 2-OH 15:0, with 2-OH 15:0 as predominant hydroxylated fatty acid. W-51T and W-52 were almost identical with respect to their phenotypic including the majority of the chemotaxonomic properties, identical in their 16S rRNA sequences, and showed 86% DNA-DNA similarity. Both strains were able to reduce nitrate and on the basis of further physiological features, a clear differentiation from all other Novosphingobium species was possible. The DNA-DNA similarities of W-51T to the type strains of N. subterraneum, N. aromaticivorans, and N. capsulatum were below 56%. For these reasons, it is proposed to create a new species with the name Novosphingobium hassiacum sp. nov.

Genome economization in the endosymbiont of the wood roach Cryptocercus punctulatus due to drastic loss of amino acid synthesis capabilities.

Cockroaches (Blattaria: Dictyoptera) harbor the endosymbiont Blattabacterium sp. in their abdominal fat body. This endosymbiont is involved in nitrogen recycling and amino acid provision to its host. In this study, the genome of Blattabacterium sp. of Cryptocercus punctulatus (BCpu) was sequenced and compared with those of the symbionts of Blattella germanica and Periplaneta americana, BBge and BPam, respectively. The BCpu genome consists of a chromosome of 605.7 kb and a plasmid of 3.8 kb and is therefore approximately 31 kb smaller than the other two aforementioned genomes. The size reduction is due to the loss of 55 genes, 23 of which belong to biosynthetic pathways for amino acids. The pathways for the production of tryptophan, leucine, isoleucine/threonine/valine, methionine, and cysteine have been completely lost. Additionally, the genes for the enzymes catalyzing the last steps of arginine and lysine biosynthesis, argH and lysA, were found to be missing and pseudogenized, respectively. These gene losses render BCpu auxotrophic for nine amino acids more than those corresponding to BBge and BPam. BCpu has also lost capacities for sulfate reduction, production of heme groups, as well as genes for several other unlinked metabolic processes, and genes present in BBge and BPam in duplicates. Amino acids and cofactors that are not synthesized by BCpu are either produced in abundance by hindgut microbiota or are provisioned via a copious diet of dampwood colonized by putrefying microbiota, supplying host and Blattabacterium symbiont with the necessary nutrients and thus permitting genome economization of BCpu.

Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome.

A novel enzyme, RA.04, belonging to the alpha-amylase family was obtained after expression of metagenomic DNA from rumen fluid (Ferrer et al.: Environ. Microbiol. 2005, 7, 1996-2010). The purified RA.04 has a tetrameric structure (280 kDa) and exhibited maximum activity (5000 U/mg protein) at 70 degrees C and was active within an unusually broad pH range from 5.5 to 9.0. It maintained 80% activity at pH 5.0 and 9.5 and 75 degrees C. The enzyme hydrolyzed alpha-D-(1,4) bonds 13-fold faster than alpha-D-(1,6) bonds to yield maltose and glucose as the main products, and it exhibited transglycosylation activity. Its preferred substrates, in the descending order, were maltooligosaccharides (C3-C7), cyclomaltoheptaose (beta-CD), cyclomaltohexaose (alpha-CD), cyclomaltooctaose (gamma-CD), soluble starch, amylose, pullulan and amylopectin. The biochemical properties and amino acid sequence alignments suggested that this enzyme is a cyclomaltodextrinase. However, despite the similarity in the catalytic module (with Glu359 and Asp331 being the catalytic nucleophile and substrate-binding residues, respectively), the enzyme bears a shorter N-terminal domain that may keep the active site more accessible for both starch and pullulan, compared to the other known CDases. Moreover, RA.04 lacks the well-conserved N-terminal Trp responsible for the substrate preference typical of CDases/MAases/PNases, suggesting a new residue is implicated in the preference for cyclic maltooligosaccharides. This study has demonstrated the usefulness of a metagenomic approach to gain novel debranching enzymes, important for the bread/food industries, from microbial environments with a high rate of plant polymer turnover, exemplified by the cow rumen.

Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora.

A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.

Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes.

The group of planctomycetes represents a separate line of descent within the domain Bacteria. Two phylum-specific 16S rRNA-targeted oligonucleotide probes for planctomycetes have been designed, optimized for in situ hybridization and used in different habitats to detect members of the group in situ. The probes, named PLA46 and PLA886, are targeting all or nearly all members of the planctomycete line of descent. Planctomycetes could be detected in almost all samples examined, e.g. a brackish water lagoon, activated sludge, and other wastewater habitats. In situ probing revealed quite uniform morphology and spatial arrangement of the detected cells but profound differences in abundance ranging from less than 0.1% to several percentage of the total cells. Single coccoid cells with diameters between 1 and 2.5 microm were dominating in most samples with the exception of the lagoon, in which rosettes of pear-shaped cells were abundant. The planctomycetes showed generally no hybridization signals with the bacterial probe EUB338, which is in accordance with base changes in their 165 rRNA sequences. A discrete ultrastructure of planctomycete cells was suggested by double staining with rRNA-targeted probes and the DNA-binding dye 4',6-diamidino-2-phenylindole (DAPI). The probe-conferred fluorescence was distributed in a ring-shaped manner around a central DAPI spot. The two probes developed extend the existing set of group-specific rRNA-targeted probes and help to elucidate the basic composition of bacterial communities in a first step of differential analysis. In situ hybridization of environmental samples indicated widespread presence of planctomycetes in different ecosystems.

Chelatobacter heintzii (Auling et al. 1993) is a later subjective synonym of Aminobacter aminovorans (Urakami et al. 1992).

Chelatobacter heintzii, which was described as a nitrilotriacetate-utilizing organism, was re-investigated in order to clarify its taxonomic position. On the basis of 16S rDNA sequence comparisons, it is obvious that this species clusters phylogenetically with species of the genus Aminobacter. The results of investigations of the fatty acid patterns, polar lipid profiles, polyamine patterns and quinone system supported this placement. The substrate-utilization profiles and fatty acid patterns of four strains (belonging to two different genomovars) revealed homogeneous results and showed high levels of similarity to Aminobacter aminovorans. DNA-DNA similarity studies confirmed that both genomovars of Chelatobacter heintzii belong to Aminobacter aminovorans. It could be shown that all species of this group are highly interrelated. On the basis of these data and previously published results, it is obvious that Chelatobacter heintzii is a later subjective synonym of Aminobacter aminovorans.

Chryseobacterium defluvii sp. nov., isolated from wastewater.

A Gram-negative, rod-shaped, non-spore-forming, yellow-pigmented bacterium (strain B2T) isolated from wastewater of a sequence batch reactor showing enhanced phosphorus removal was investigated to determine its taxonomic status. Complete 16S rRNA gene sequence analysis indicated that the organism should be placed in the genus Chryseobacterium. The strain contained a polyamine pattern with sym-homospermidine as the major compound, menaquinone MK-6 as the predominant menaquinone and ai-C15:0, i-C15:0 and C16:1 as the major fatty acids. Phosphatidylethanolamine and several unidentified lipids were detected in the polar lipid profile. Phylogenetically, strain B2T was most closely related to Chryseobacterium indoltheticum and Chryseobacterium gleum (96.2 and 95-9% 16S rRNA gene sequence similarity, respectively). The phylogenetic distance from any validly described species within the genus Chryseobacterium, as indicated from 16S rRNA gene sequence similarities, and its phenotypic properties demonstrate that strain B2T represents a novel species, for which the name Chryseobacterium defluvii sp. nov. is proposed; the type strain is B2T (=DSM 14219T =CIP 107207T).

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes.

Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G + C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol 2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0.1 to 650 mU (mg protein)-1. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains.