Molecular and Microbiological Insights on the Enrichment Procedures for the Isolation of Petroleum Degrading Bacteria and Fungi

Autochthonous bioaugmentation, by exploiting the indigenous microorganisms of the contaminated environment to be treated, can represent a successful bioremediation strategy. In this perspective, we have assessed by molecular methods the evolution of bacterial and fungal communities during the selective enrichment on different pollutants of a soil strongly polluted by mixtures of aliphatic and polycyclic hydrocarbons. Three consecutive enrichments were carried out on soil samples from different soil depths (0–1, 1–2, 2–3 m), and analyzed at each step by means of high-throughput sequencing of bacterial and fungal amplicons biomarkers. At the end of the enrichments, bacterial and fungal contaminants degrading strains were isolated and identified in order to (i) compare the composition of enriched communities by culture-dependent and culture-independent molecular methods and to (ii) obtain a collection of hydrocarbon degrading microorganisms potentially exploitable for soil bioremediation. Molecular results highlighted that for both bacteria and fungi the pollutant had a partial shaping effect on the enriched communities, with paraffin creating distinct enriched bacterial community from oil, and polycyclic aromatic hydrocarbons generally overlapping; interestingly neither the soil depth or the enrichment step had significant effects on the composition of the final enriched communities. Molecular analyses well-agreed with culture-dependent analyses in terms of most abundant microbial genera. A total of 95 bacterial and 94 fungal strains were isolated after selective enrichment procedure on different pollutants. On the whole, isolated bacteria where manly ascribed to Pseudomonas genus followed by Sphingobacterium, Bacillus, Stenothrophomonas, Achromobacter, and Serratia. As for fungi, Fusarium was the most abundant genus followed by Trichoderma and Aspergillus. The species comprising more isolates, such as Pseudomonas putida, Achromobacter xylosoxidans and Ochromobactrum anthropi for bacteria, Fusarium oxysporum and Fusarium solani for fungi, were also the dominant OTUs assessed in Illumina

Evaluation of the ISO Standard 11063 DNA extraction procedure for assessing soil microbial abundance and community structure

Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities

Vers une optimisation de la procédure d’extraction d’ADN des sols pour caractériser la diversité microbienne tellurique par le pyroséquençage des gènes ribosomiques

EASPEGenoSolEcolDurCT3Depuis plusieurs années, les communautés microbiennes du sol sont étudiées au travers de leur ADN. Le mode d’extraction de l’ADN du sol revêt donc une importance capitale pour décrire la biodiversité microbienne. A ce jour, de nombreuses procédures d’extraction d’ADN des sols sont disponibles et certaines sont mêmes standardisées (norme ISO 11063). Ces différentes techniques sont généralement basées sur une étape de lyse physique et de lyse chimique et sont surtout adaptées aux outils moléculaires de type qPCR et empreintes moléculaires. Le développement récent des techniques de séquençage massif parallèle (pyroséquençage 454), qui permettent d’aborder des inventaires taxonomiques précis et représentatifs, nous conduit à revisiter les biais associés à ces procédures d’extraction d’ADN. L’objectif de cette étude est de définir le protocole d’extraction d’ADN du sol le plus approprié pour accéder au maximum de diversité microbienne tellurique. Dans ce contexte, différentes procédures sont testées (dont la norme ISO) sur 5 sols différents de par leurs propriétés physicochimiques et leur mode d’usage. La qualité des ADN extraits selon ces différents protocoles est évaluée en termes de rendement d’extraction, d’abondance (biomasse moléculaire par qPCR) et de diversité (pyroséquençage 454 des gènes ribosomiques) des communautés bactériennes et fongiques. L’analyse des différents résultats permet de définir le protocole le mieux adapté pour accéder à la meilleure représentativité de la diversité des communautés microbiennes telluriques

Apport des nouvelles générations de séquençage pour accéder à la diversité des communautés microbiennes du sol : nécessité d’un ‘pipeline’ bio-informatique pour les biologistes

National audienc

Apport des nouvelles générations de séquençage pour accéder à la diversité et la richesse des communautés microbiennes de sols issus d'échantillonnage de grande ampleur

Une présentation orale a été faite lors de ce congrèsEASPEGenoSolEcolDurCT3 (vu ST)La diversité microbienne d’un sol (que l’on estime entre 105 et 106 espèces différentes par gramme de sol) est difficile à caractériser. Cependant, les techniques de séquençage haut-débit comme le pyroséquençage permettent maintenant d'étudier la diversité des communautés microbiennes du sol en se basant sur un marqueur phylogénétique (comme l'ADNr 16S ou 18S). De premières études ont déjà été réalisées avec cette technique afin d’aborder la diversité bactérienne des sols et cette dernière est maintenant unanimement reconnue pour sa pertinence et ses potentialités très importantes. Toutefois, l’écologie microbienne du sol commence à se tourner vers l'étude d'échantillonnages de grande envergure spatiale (Réseau de Mesure de la Qualité des Sols qui représente plus de 2200 sols à l'échelle du territoire français) afin de mieux hiérarchiser les processus et paramètres impliqués dans l'assemblage de ces communautés. Ce caractère massif, ainsi que les caractéristiques inhérentes aux séquences obtenues par la technique de pyroséquençage requièrent le développement d’outils bioinformatiques adaptés, optimisés et évalués, afin d’analyser rapidement et efficacement ce type de données. L’objectif final est de coupler, sur un grand nombre d’échantillons, cette approche avec des mesures d’activités et de faire le lien entre la diversité microbienne tellurique et l’aptitude des sols à rendre des services. Les premiers résultats (méthodologiques et biologiques) sur un nombre restreint de sols seront décrits et présentés

Evaluation of the ISO standard 11063 “Soil DNA Extraction Procedure” for Assessing Microbial Abundance and Community Structure

EASPEEcolDurGenoSolCT3For the last two decades, soil microbial diversity studies have been dependent upon the extraction and characterization of soil DNA. Therefore, the DNA extraction procedure has become a critical step in describing soil microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures which affect the assessments of diversity and community composition. For that reason, a standardized soil DNA extraction method (ISO 11063) was developed. However, this standard has only been optimized for examining soil bacteria, and not for other soil microbes (archaea and fungi). Similar to other extraction procedures, this standard protocol relies on different physical and chemical lysis steps, and is mainly adapted to molecular tools such as qPCR and molecular fingerprinting techniques. The recent development of massively parallel sequencing technologies which permit more representative and accurate taxonomical inventories, has instigated a reassessment of the biases associated with the DNA extraction procedure. Therefore, the aim of this study was to assess the most appropriate soil DNA extraction procedure for studying soil diversity. In this context, three different procedures were tested (the ISO standard, an optimization of the ISO procedure (ISOm), and a custom procedure (GnS-GII)) on five soils of differing land use and physicochemical properties. The quality of the DNA extracted according to these different procedures was evaluated in terms of yield, microbial abundance and diversity. These analyses are being confirmed by a 454 pyrosequencing analysis. Results clearly showed that DNA yield was higher using the ISOm and GnS-GII procedures compared to the ISO. Moreover, the abundance of bacteria, archaea and fungi were found to be lower with the ISO extraction method. Bacterial diversity patterns exhibited similar results, with a strong impact of soil pH on diversity profiles. Fungal community structure was strongly affected by the DNA extraction protocol, with soil type having only a secondary effect for the ISOm and GnS-GII methods. Archaeal diversity patterns were influenced both by soil physicochemical properties and the DNA extraction procedure. These results indicate that the ISO standard is a suitable extraction procedure for studies focusing on soil bacteria. However, for larger scale studies which aim to examine bacteria, archaea and fungi the standardized procedure would not be appropriate. Indeed, the ISOm procedure, which is an improvement of the standard, produces results which are more reproducible and are more representative of soil microbial communitie

Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure

International audienceThree soil DNA extraction procedures (homemade protocols and commercial kit) varying in their practicability were applied to contrasting soils to evaluate their efficiency in recovering: (i) soil DNA and (ii) bacterial diversity estimated by 16S rDNA pyrosequencing. Significant differences in DNA yield were systematically observed between tested procedures. For certain soils, 10 times more DNA was recovered with one protocol than with the others. About 15 000 sequences of 16S rDNA were obtained for each sample which were clustered to draw rarefaction curves. These curves, as well as the PCA ordination of community composition based on OTU clustering, did not reveal any significant difference between procedures. Nevertheless, significant differences between procedures were highlighted by the taxonomic identification of sequences obtained at the phylum to genus levels. Depending on the soil, differences in the number of genera detected ranged from 1% to 26% between the most and least efficient procedures, mainly due to a poorer capacity to recover populations belonging to Actinobacteria, Firmicutes or Crenarchaeota. This study enabled us to rank the relative efficiencies of protocols for their recovery of soil molecular microbial biomass and bacterial diversity and to help choosing an appropriate soil DNA extraction procedure adapted to novel sequencing technologies

Biogeographical patterns of soil bacterial diversity at the scale of France

EASPEBIOMEGENOSOLBiogeographical patterns of soil bacterial diversity at the scale of France. 16. International Symposium on Microbial Ecology - ISME1

Shifts in microbial diversity through land use intensity as drivers of carbon mineralization in soil.

10 pagesInternational audienceLand use practices alter the biomass and structure of soil microbial communities. However, the impact of land management intensity on soil microbial diversity (i.e. richness and evenness) and consequences for functioning is still poorly understood. Here, we addressed this question by coupling molecular characterization of microbial diversity with measurements of carbon (C) mineralization in soils obtained from three locations across Europe, each representing a gradient of land management intensity under different soil and environmental conditions. Bacterial and fungal diversity were characterized by high throughput sequencing of ribosomal genes. Carbon cycling activities (i.e., mineralization of autochthonous soil organic matter, mineralization of allochthonous plant residues) were measured by quantifying 12C- and 13C-CO2 release after soils had been amended, or not, with 13C-labelled wheat residues. Variation partitioning analysis was used to rank biological and physicochemical soil parameters according to their relative contribution to these activities. Across all three locations, microbial diversity was greatest at intermediate levels of land use intensity, indicating that optimal management of soil microbial diversity might not be achieved under the least intensive agriculture. Microbial richness was the best predictor of the C-cycling activities, with bacterial and fungal richness explaining 32.2 and 17% of the intensity of autochthonous soil organic matter mineralization; and fungal richness explaining 77% of the intensity of wheat residues mineralization. Altogether, our results provide evidence that there is scope for improvement in soil management to enhance microbial biodiversity and optimize C transformations mediated by microbial communities in soil