Microbiological versus Chemical Reductive Sulfidation: An Experimental and Theoretical Study.

Microbiological reductive sulfidation (RS) has rarely been documented, although it represents an efficient strategy for thiol formation. In this work, we reported on the sulfate-respiring bacterium Desulfovibrio sp.86 that has previously demonstrated RS activity toward the pesticide chlordecone. The purpose of this study was to assess its substrate versatility using a set of 28 carbonyls, to compare with chemical RS and to rationalize the observed trends using a dual experimental and theoretical approach. The chemical RS generally proceeds in two steps (S/O exchange using a sulfur donor like P4S10, reduction of the thione intermediate). Intriguingly, chlordecone was found to be converted into chlordecthiol following the first step. Hence, we designed a protocol and applied it to the 28 substrates to assess their propensity to be directly converted into thiols with the P4S10 treatment alone. Finally, we performed density functional theory calculations on these carbonyls and their thiocarbonyl derivatives to build a set of structural, electronic, and thermodynamic parameters. The results showed that chemical and microbiological RS probably involved two distinct mechanisms. Chemically, we observed that several carbonyls, possessing electron-withdrawing groups and/or aromatic rings, were directly transformed into thiols in the presence of P4S10. The correlation obtained with the electron affinity of the thiones led us to conclude that a probable single-electron reductive transfer occurred during the first step. We also found that Desulfovibrio sp.86 transformed a variety of aldehydes and ketones, without ever detecting thiones. No significant correlation was observed with the calculated parameters, but a relationship between aldehyde RS biotransformation and bacterial growth was observed. Differences in selectivity with chemical RS open the way for further applications in organic synthesis.

Genetic Analysis of Citrobacter sp.86 Reveals Involvement of Corrinoids in Chlordecone and Lindane Biotransformations.

Chlordecone (Kepone®) and γ-hexachlorocyclohexane (γ-HCH or lindane) have been used for decades in the French West Indies (FWI) resulting in long-term soil and water pollution. In a previous work, we have identified a new Citrobacter species (sp.86) that is able to transform chlordecone into numerous products under anaerobic conditions. No homologs to known reductive dehalogenases or other candidate genes were found in the genome sequence of Citrobacter sp.86. However, a complete anaerobic pathway for cobalamin biosynthesis was identified. In this study, we investigated whether cobalamin or intermediates of cobalamin biosynthesis was required for chlordecone microbiological transformation. For this purpose, we constructed a set of four Citrobacter sp.86 mutant strains defective in several genes belonging to the anaerobic cobalamin biosynthesis pathway. We monitored chlordecone and its transformation products (TPs) during long-term incubation in liquid cultures under anaerobic conditions. Chlordecone TPs were detected in the case of cobalamin-producing Citrobacter sp.86 wild-type strain but also in the case of mutants able to produce corrinoids devoid of lower ligand. In contrast, mutants unable to insert the cobalt atom in precorrin-2 did not induce any transformation of chlordecone. In addition, it was found that lindane, previously shown to be anaerobically transformed by Citrobacter freundii without evidence of a mechanism, was also degraded in the presence of the wild-type strain of Citrobacter sp.86. The lindane degradation abilities of the various Citrobacter sp.86 mutant strains paralleled chlordecone transformation. The present study shows the involvement of cobalt-containing corrinoids in the microbial degradation of chlorinated compounds with different chemical structures. Their increased production in contaminated environments could accelerate the decontamination processes.

Transformation of the recalcitrant pesticide chlordecone by Desulfovibrio sp.86 with a switch from ring-opening dechlorination to reductive sulfidation activity.

The insecticide chlordecone has been used in the French West Indies for decades, resulting in long term pollution, human health problems and social crisis. In addition to bacterial consortia and Citrobacter sp.86 previously described to transform chlordecone into three families of transformation products (A: hydrochlordecones, B: polychloroindenes and C: polychloroindenecarboxylic acids), another bacterium Desulfovibrio sp.86, showing the same abilities has been isolated and its genome was sequenced. Ring-opening dechlorination, leading to A, B and C families, was observed as previously described. Changing operating conditions in the presence of chlordecone gave rise to the formation of an unknown sulfur-containing transformation product instead of the aforementioned ones. Its structural elucidation enabled to conclude to a thiol derivative, which corresponds to an undocumented bacterial reductive sulfidation. Microbial experiments pointed out that the chlordecone thiol derivative was observed in anaerobiosis, and required the presence of an electron acceptor containing sulfur or hydrogen sulfide, in a confined atmosphere. It seems that this new reaction is also active on hydrochlordecones, as the 10-monohydrochlordecone A1 was transformed the same way. Moreover, the chlordecone thiol derivative called F1 was detected in several chlordecone contaminated mangrove bed sediments from Martinique Island, highlighting the environmental relevance of these results.

In situ metabolic activities of uncultivated Ferrovum sp. CARN8 evidenced by metatranscriptomic analysis.

Amongst iron-oxidizing bacteria playing a key role in the natural attenuation of arsenic in acid mine drainages (AMDs), members of the Ferrovum genus were identified in mine effluent or water treatment plants, and were shown to dominate biogenic precipitates in field pilot experiments. In order to address the question of the in situ activity of the uncultivated Ferrovum sp. CARN8 strain in the Carnoulès AMD, we assembled its genome using metagenomic and metatranscriptomic sequences and we determined standardized expression values for protein-encoding genes. Our results showed that this microorganism was indeed metabolically active and allowed us to sketch out its metabolic activity in its natural environment. Expression of genes related to the respiratory chain and carbon fixation suggests aerobic energy production coupled to ferrous iron oxidation and chemolithoautotrophic growth. Notwithstanding the presence of nitrogenase genes in its genome, expression data also indicated that Ferrovum sp. CARN8 relied on ammonium import rather than nitrogen fixation. The expression of flagellum and chemotaxis genes hints that at least a proportion of this strain population was motile. Finally, apart from some genes related to metal resistance showing surprisingly low expression values, genes involved in stress response were well expressed as expected in AMDs.

Natural Chlordecone Degradation Revealed by Numerous Transformation Products Characterized in Key French West Indies Environmental Compartments.

Production and use of the insecticide chlordecone has caused long-term environmental pollution in the James River area and the French West Indies (FWI) that has resulted in acute human-health problems and a social crisis. High levels of chlordecone in FWI soils, even after its ban decades ago, and the absence of detection of transformation products (TPs), have suggested that chlordecone is virtually nonbiodegradable in the environment. Here, we investigated laboratory biodegradation, consisting of bacterial liquid cultures and microcosms inoculated with FWI soils, using a dual nontargeted GC-MS and LC-HRMS approach. In addition to previously reported, partly characterized hydrochlordecones and polychloroindenes (families A and B), we discovered 14 new chlordecone TPs, assigned to four families (B, C, D, and E). Organic synthesis and NMR analyses allowed us to achieve the complete structural elucidation of 19 TPs. Members of TP families A, B, C, and E were detected in soil, sediment, and water samples from Martinique and include 17 TPs not initially found in commercial chlordecone formulations. 2,4,5,6,7-Pentachloroindene was the most prominent TP, with levels similar to those of chlordecone. Overall, our results clearly show that chlordecone pollution extends beyond the parent chlordecone molecule and includes a considerable number of previously undetected TPs. Structural diversity of the identified TPs illustrates the complexity of chlordecone degradation in the environment and raises the possibility of extensive worldwide pollution of soil and aquatic ecosystems by chlordecone TPs.

Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters.

The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 Å resolution and revealed a modified α/ß hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.

Distinct Carbon Isotope Fractionation Signatures during Biotic and Abiotic Reductive Transformation of Chlordecone.

Chlordecone is a synthetic organochlorine pesticide, extensively used in banana plantations of the French West Indies from 1972 to 1993. Due to its environmental persistence and bioaccumulation, it has dramatic public health and socio-economic impact. Here we describe a method for carbon-directed compound specific isotope analysis (CSIA) for chlordecone and apply it to monitor biotic and abiotic reductive transformation reactions, selected on the basis of their distinct product profiles (polychloroindenes versus lower chlorinated hydrochlordecones). Significant carbon isotopic enrichments were observed for all microbially mediated transformations (εbulk = -6.8‰ with a Citrobacter strain and εbulk = -4.6‰ with a bacterial consortium) and for two abiotic transformations (εbulk = -4.1‰ with zerovalent iron and εbulk = -2.6‰ with sodium sulfide and vitamin B12). The reaction with titanium(III) citrate and vitamin B12, which shows the product profile most similar to that observed in biotic transformation, led to low carbon isotope enrichment (εbulk =-0.8‰). The CSIA protocol was also applied on representative chlordecone formulations previously used in the French West Indies, giving similar chlordecone δ13C values from -31.1 ± 0.2‰ to -34.2 ± 0.2‰ for all studied samples. This allows the in situ application of CSIA for the assessment of chlordecone persistence.

Microbial Degradation of a Recalcitrant Pesticide: Chlordecone.

Chlordecone (Kepone®) is a synthetic organochlorine insecticide (C10Cl10O) used worldwide mostly during the 1970 and 1980s. Its intensive application in the French West Indies to control the banana black weevil Cosmopolites sordidus led to a massive environmental pollution. Persistence of chlordecone in soils and water for numerous decades even centuries causes global public health and socio-economic concerns. In order to investigate the biodegradability of chlordecone, microbial enrichment cultures from soils contaminated by chlordecone or other organochlorines and from sludge of a wastewater treatment plant have been conducted. Different experimental procedures including original microcosms were carried out anaerobically over long periods of time. GC-MS monitoring resulted in the detection of chlorinated derivatives in several cultures, consistent with chlordecone biotransformation. More interestingly, disappearance of chlordecone (50 µg/mL) in two bacterial consortia was concomitant with the accumulation of a major metabolite of formula C9Cl5H3 (named B1) as well as two minor metabolites C10Cl9HO (named A1) and C9Cl4H4 (named B3). Finally, we report the isolation and the complete genomic sequences of two new Citrobacter isolates, closely related to Citrobacter amalonaticus, and that were capable of reproducing chlordecone transformation. Further characterization of these Citrobacter strains should yield deeper insights into the mechanisms involved in this transformation process.

Reconstructing rare soil microbial genomes using in situ enrichments and metagenomics.

Despite extensive direct sequencing efforts and advanced analytical tools, reconstructing microbial genomes from soil using metagenomics have been challenging due to the tremendous diversity and relatively uniform distribution of genomes found in this system. Here we used enrichment techniques in an attempt to decrease the complexity of a soil microbiome prior to sequencing by submitting it to a range of physical and chemical stresses in 23 separate microcosms for 4 months. The metagenomic analysis of these microcosms at the end of the treatment yielded 540 Mb of assembly using standard de novo assembly techniques (a total of 559,555 genes and 29,176 functions), from which we could recover novel bacterial genomes, plasmids and phages. The recovered genomes belonged to Leifsonia (n = 2), Rhodanobacter (n = 5), Acidobacteria (n = 2), Sporolactobacillus (n = 2, novel nitrogen fixing taxon), Ktedonobacter (n = 1, second representative of the family Ktedonobacteraceae), Streptomyces (n = 3, novel polyketide synthase modules), and Burkholderia (n = 2, includes mega-plasmids conferring mercury resistance). Assembled genomes averaged to 5.9 Mb, with relative abundances ranging from rare (<0.0001%) to relatively abundant (>0.01%) in the original soil microbiome. Furthermore, we detected them in samples collected from geographically distant locations, particularly more in temperate soils compared to samples originating from high-latitude soils and deserts. To the best of our knowledge, this study is the first successful attempt to assemble multiple bacterial genomes directly from a soil sample. Our findings demonstrate that developing pertinent enrichment conditions can stimulate environmental genomic discoveries that would have been impossible to achieve with canonical approaches that focus solely upon post-sequencing data treatment.

16S rRNA and As-Related Functional Diversity: Contrasting Fingerprints in Arsenic-Rich Sediments from an Acid Mine Drainage.

To gain an in-depth insight into the diversity and the distribution of genes under the particular evolutionary pressure of an arsenic-rich acid mine drainage (AMD), the genes involved in bacterial arsenic detoxification (arsB, ACR3) and arsenite oxidation (aioA) were investigated in sediment from Carnoulès (France), in parallel to the diversity and global distribution of the metabolically active bacteria. The metabolically active bacteria were affiliated mainly to AMD specialists, i.e., organisms detected in or isolated from AMDs throughout the world. They included mainly Acidobacteria and the non-affiliated "Candidatus Fodinabacter communificans," as well as Thiomonas and Acidithiobacillus spp., Actinobacteria, and unclassified Gammaproteobacteria. The distribution range of these organisms suggested that they show niche conservatism. Sixteen types of deduced protein sequences of arsenite transporters (5 ArsB and 11 Acr3p) were detected, whereas a single type of arsenite oxidase (AioA) was found. Our data suggested that at Carnoulès, the aioA gene was more recent than those encoding arsenite transporters and subjected to a different molecular evolution. In contrast to the 16S ribosomal RNA (16S rRNA) genes associated with AMD environments worldwide, the functional genes aioA, ACR3, and to a lesser extent arsB, were either novel or specific to Carnoulès, raising the question as to whether these functional genes are specific to high concentrations of arsenic, AMD-specific, or site-specific.

Growth of nitrite-oxidizing bacteria by aerobic hydrogen oxidation.

The bacterial oxidation of nitrite to nitrate is a key process of the biogeochemical nitrogen cycle. Nitrite-oxidizing bacteria are considered a highly specialized functional group, which depends on the supply of nitrite from other microorganisms and whose distribution strictly correlates with nitrification in the environment and in wastewater treatment plants. On the basis of genomics, physiological experiments, and single-cell analyses, we show that Nitrospira moscoviensis, which represents a widely distributed lineage of nitrite-oxidizing bacteria, has the genetic inventory to utilize hydrogen (H2) as an alternative energy source for aerobic respiration and grows on H2 without nitrite. CO2 fixation occurred with H2 as the sole electron donor. Our results demonstrate a chemolithoautotrophic lifestyle of nitrite-oxidizing bacteria outside the nitrogen cycle, suggesting greater ecological flexibility than previously assumed.

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon "Ca. Nitrosotenuis uzonensis" representing a clade globally distributed in thermal habitats.

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation.

Gene capture coupled to high-throughput sequencing as a strategy for targeted metagenome exploration.

Next-generation sequencing (NGS) allows faster acquisition of metagenomic data, but complete exploration of complex ecosystems is hindered by the extraordinary diversity of microorganisms. To reduce the environmental complexity, we created an innovative solution hybrid selection (SHS) method that is combined with NGS to characterize large DNA fragments harbouring biomarkers of interest. The quality of enrichment was evaluated after fragments containing the methyl coenzyme M reductase subunit A gene (mcrA), the biomarker of methanogenesis, were captured from a Methanosarcina strain and a metagenomic sample from a meromictic lake. The methanogen diversity was compared with direct metagenome and mcrA-based amplicon pyrosequencing strategies. The SHS approach resulted in the capture of DNA fragments up to 2.5 kb with an enrichment efficiency between 41 and 100%, depending on the sample complexity. Compared with direct metagenome and amplicons sequencing, SHS detected broader mcrA diversity, and it allowed efficient sampling of the rare biosphere and unknown sequences. In contrast to amplicon-based strategies, SHS is less biased and GC independent, and it recovered complete biomarker sequences in addition to conserved regions. Because this method can also isolate the regions flanking the target sequences, it could facilitate operon reconstructions.

Characterization of rhizosphere prokaryotic diversity in a horizontal subsurface flow constructed wetland using a PCR cloning-sequencing based approach.

Performance of biological wastewater treatment systems may be related to the composition and activity of microbial populations they contain. However, little information is known regarding microbial community inhabiting these ecosystems. The purpose of this study was to investigate archaeal and bacterial diversity, using cultivation-independent molecular techniques, in a constructed wetland receiving domestic wastewater. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by PCR using primers specific for archaeal and bacterial domains. A high microbial diversity was detected. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 31.3 % of the OTUs, followed by the Bacteroidetes (14.8 %), Planctomycetales (13.8 %), Actinobacteria (12 %), and Chloroflexi (8.2 %). Sequences affiliated with minor phylogenetic divisions such as the TM7, Nitrospira, OP10, and BRC1 are represented by <6 % of total OTUs. The Archaea domain was represented by the Thaumarchaeota phylum dominated by the Candidatus Nitrososphaera genus.

The structure and spatio-temporal distribution of the Archaea in a horizontal subsurface flow constructed wetland.

In this study, archaeal community structure and temporal dynamics were monitored, using 16S rRNA clone libraries construction from a horizontal subsurface flow constructed wetland. Phylogenetic assignation of 1026 16S rRNA gene sequences shows that 96.2% of the total operational taxonomic units (OTUs) were affiliated with Thaumarchaeota, a newly proposed archaeal phylum and 3.7% with unclassified Archaea. Among the total sequences, 42% and 40.2% were affiliated with Candidatus Nitrososphaera and unclassified Nitrosopumilus respectively with more than 99% similarity. Results suggest that several dominant and active nitrifiers may benefit from the micro-aerobic conditions around the reed roots to perform ammonia oxidation. The archaeal diversity detected in the rhizosphere zone is clearly different from that detected in the bottom basin. This engineered habitat revealed the reed root and the water composition effects on the archaeal diversity.

Nitrification expanded: discovery, physiology and genomics of a nitrite-oxidizing bacterium from the phylum Chloroflexi.

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, a major process of the biogeochemical nitrogen cycle, but the recognized diversity of this guild is surprisingly low and only two bacterial phyla contain known NOB. Here, we report on the discovery of a chemolithoautotrophic nitrite oxidizer that belongs to the widespread phylum Chloroflexi not previously known to contain any nitrifying organism. This organism, named Nitrolancetus hollandicus, was isolated from a nitrifying reactor. Its tolerance to a broad temperature range (25-63 °C) and low affinity for nitrite (K(s)=1 mM), a complex layered cell envelope that stains Gram positive, and uncommon membrane lipids composed of 1,2-diols distinguish N. hollandicus from all other known nitrite oxidizers. N. hollandicus grows on nitrite and CO(2), and is able to use formate as a source of energy and carbon. Genome sequencing and analysis of N. hollandicus revealed the presence of all genes required for CO(2) fixation by the Calvin cycle and a nitrite oxidoreductase (NXR) similar to the NXR forms of the proteobacterial nitrite oxidizers, Nitrobacter and Nitrococcus. Comparative genomic analysis of the nxr loci unexpectedly indicated functionally important lateral gene transfer events between Nitrolancetus and other NOB carrying a cytoplasmic NXR, suggesting that horizontal transfer of the NXR module was a major driver for the spread of the capability to gain energy from nitrite oxidation during bacterial evolution. The surprising discovery of N. hollandicus significantly extends the known diversity of nitrifying organisms and likely will have implications for future research on nitrification in natural and engineered ecosystems.

Structure, fluctuation and magnitude of a natural grassland soil metagenome.

The soil ecosystem is critical for human health, affecting aspects of the environment from key agricultural and edaphic parameters to critical influence on climate change. Soil has more unknown biodiversity than any other ecosystem. We have applied diverse DNA extraction methods coupled with high throughput pyrosequencing to explore 4.88 × 10(9) bp of metagenomic sequence data from the longest continually studied soil environment (Park Grass experiment at Rothamsted Research in the UK). Results emphasize important DNA extraction biases and unexpectedly low seasonal and vertical soil metagenomic functional class variations. Clustering-based subsystems and carbohydrate metabolism had the largest quantity of annotated reads assigned although <50% of reads were assigned at an E value cutoff of 10(-5). In addition, with the more detailed subsystems, cAMP signaling in bacteria (3.24±0.27% of the annotated reads) and the Ton and Tol transport systems (1.69±0.11%) were relatively highly represented. The most highly represented genome from the database was that for a Bradyrhizobium species. The metagenomic variance created by integrating natural and methodological fluctuations represents a global picture of the Rothamsted soil metagenome that can be used for specific questions and future inter-environmental metagenomic comparisons. However, only 1% of annotated sequences correspond to already sequenced genomes at 96% similarity and E values of <10(-5), thus, considerable genomic reconstructions efforts still have to be performed.

Betaproteobacteria dominance and diversity shifts in the bacterial community of a PAH-contaminated soil exposed to phenanthrene.

In this study, the PAH-degrading bacteria of a constructed wetland collecting road runoff has been studied through DNA stable isotope probing. Microcosms were spiked with (13)C-phenanthrene at 34 or 337 ppm, and bacterial diversity was monitored over a 14-day period. At 337 ppm, PAH degraders became dominated after 5 days by Betaproteobacteria, including novel Acidovorax, Rhodoferax and Hydrogenophaga members, and unknown bacteria related to Rhodocyclaceae. The prevalence of Betaproteobacteria was further demonstrated by phylum-specific quantitative PCR, and was correlated with a burst of phenanthrene mineralization. Striking shifts in the population of degraders were observed after most of the phenanthrene had been removed. Soil exposed to 34 ppm phenanthrene showed a similar population of degraders, albeit only after 14 days. Results demonstrate that specific Betaproteobacteria are involved in the main response to soil PAH contamination, and illustrate the potential of SIP approaches to investigate PAH biodegradation in soil.