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Tuesday 21 August 1 PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August PS16 – Soil Microbiology and Heterogeneity 076B Soil persistent Escherichia coli Florence Abram*1, Nicole Clarke1, John Murphy1, Emma Moynihan2, Fiona Brennan2 1NUI, Galway, Ireland, 2Teagasc, Ireland An understanding of the survival capacity of Escherichia coli in soil is critical for the evaluation of its role as an indicator of environmental faecal pollution. Recent reports that E. coli can become long-term residents in Irish soils have raised the question of how the organism survives and competes for ecological niche in the inhospitable soil environment. This study examined the genetic diversity and the phenotypic characteristics of E. coli in Irish soil. Molecular analysis determined the genetic diversity of a collection of environmentally persistent E. coli, while phenotypic methods aimed at evaluating the metabolic capacity as well as the soil survival capacity and the environmental fitness of the soil isolates. Our results demonstrate that Irish soil isolates do not form a single genetic grouping but that multiple clonal groups are capable of surviving and proliferating in this environment. Phenotypic analyses indicated that soil E. coli possessed a wide metabolic flexibility at low temperature, probably rendered possible by the redirection of protein expression in response to cold exposure as revealed by proteomics. In addition, when introduced in soil microcosms at low temperature, environmentally persistent E. coli displayed a greater survival capacity compared to clinical isolates. Overall, these findings suggest that E. coli has the ability to become naturalized in Irish soils, which has serious implications for its use as an indicator of faecal pollution in the environment. 077B Diversity and composition of nitrogen fixing communities in Argentinean soils as affected by agricultural practices O. Mario Aguilar*1, Monica Collavino2, James Tripp3, Priscila Calderoli2, María Laura Vidoz4, Mariano Donato5, Jonathan P. Zehr3 1IBBM-CONICET, Universidad Nacional de La Plata, Facultad Ciencias Exactas, Argentina, 2Universidad Nacional de La Plata, Facultad Ciencias Exactas, Argentina, 3University of California, USA, 4Universidad del Nordeste, Argentina, 5Universidad Nacional de La Plata, Facultad Ciencias Naturales y Museo, Argentina Microbial community may have significant impacts on important soil processes; however, there is still limited information about the effect of crop management on the diversity and composition of soil microbial community. In this study, we examined the diazotroph community in Argentinean soils subjected to different agricultural practices (crop rotation with nutrient amendment, monocropping without nutrient reposition, and non-cultivated soil), using nifH gene as a molecular marker. DNA was extracted from soil samples collected at four sites in Argentina: Pergamino (province of Buenos Aires), Viale (province of Entre Ríos), Bengolea and Monte Buey (province of Córdoba). The diversity and composition of diazotrophic community were analyzed by deep pyrosequencing of nifH sequences, the relative abundance by qPCR whereas the active diazotroph populations were assessed by nifH mRNA analysis after reverse transcription. About 183,000 reads were processed and short sequences, putative chimeras and frameshifts were removed. The resulting ARB database consisting of 87020 reads was grouped into 1558 OTUs assuming a level of dissimilarity of 0.02% at amino acid sequence level. Comparison of these sequences with the public nifH database, showed that the OTUs identified in Argentinean soils were distributed among the four major lineages (clusters I to IV) known to exist throughout the world in various environments. This indicates that a broad range of diversity is represented in our ARB database and in Argentinean soils. Cluster I was most highly represented in our samples, particularly subclusters 1K and 1J (42% and 20%, respectively), which contain sequences mainly belonging to the orders Rhizobiales and Burkholderiales. Different estimators were applied to assess richness and evenness of the community. Both components showed different degree of diversity, however Bengolea, which represents the sandiest soil, rendered the lowest values independently of the year and treatment. In each location of sampling, richness was in general high in cultivated treatments, except in intensive rotation treatments under corn where the diversity was significantly lower. These results suggest that the diversity of the 1 PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August nitrogen-fixing community is affected by soil type, agronomic practices and, particularly, by the crop species used in the rotation. In contrast, analysis of RNA revealed that non-cultivated (pristine) soil displayed higher diversity as compared to cultivated soils. Associations between the diazotrophic communities and treatments were searched by Principal Coordinates Analysis (PCoA) and Cluster Analysis (CA) using distance matrices constructed with the OTUs data (abundance and presence-absence). It was found in general, that different locations showed distinctive diazotrophic community. In addition, OTU abundance in each site was significantly affected by the treatment. In conclusion, these results show that diversity, composition and functionality of the diazotroph community varies among soils from different locations, as well as in soils with different agricultural practices. 078B Application of quantitative PCR methods for the analysis of a BTEX contaminated plume Christopher Allen*, Paul Flanagan, Anna Kulakova, Leonid Kulakov, Michael J Larkin Queen's University Belfast, United Kingdom In this research we studied a BTEX contaminated plume using metagenomic approaches. BTEX is a major industrial pollutant and is a significant concern in the petrochemical industries. We sampled the anaerobic zone of a contaminated area and used quantitative PCR to compare the prevalence of key catabolic genes – associated with the degradation of BTEX under both aerobic and anaerobic conditions – when compared to a BTEX-free control. We wanted to see if the relative quantity of catabolic genes detected could help understand the mechanism of contaminant biodegradation. Methods used included quantitative PCR and metagenomic 16S rRNA gene population characterisation approaches (both OTU analysis and 454 sequencing were employed). Further mass spectrometry analysis was also performed to confirm the nature of contaminants in the plume and a designated control. The research showed that the contaminated area was dominated by a limited number of eubacteria – that are established facultative anaerobe BTEX degraders. These appear to be mainly nitrate-reducing bacteria. This was expected as nitrate is also present as a significant inorganic contaminant. The quantitative PCR analysis also suggests that biodegradation is primarily anaerobic – as the metagenome was dominated by catabolic gene sequences associated with anaerobic degradation of aromatic compounds. Our data also suggest that there may be two other processes occurring on the site: i) There is evidence of horizontal gene transfer leading to, specifically, enhanced benzene degradation in the plume ii) There is also evidence that the dominant BTEX degrader has undergone significant evolutionary change over the course of the polluting event (10+ years). These observations will be discussed in more detail in the poster. BTEX is degraded in the site. The degrading population is mainly comprised of one species of facultative anaerobic bacteria, although other groups do play a significant role. 079B Bacterial nitrifying and denitrifying communities in soils with organic and conventional agricultural practices Celine Amsaleg*, Mehdi Zerarka, Anniet M Laverman, Josette Garnier University Pierre and Marie Curie, France Elevated nitrogen concentrations and green-house gas emissions (mainly N2O) in and from soils are of environmental concern. The Seine River basin (France) dominated by areas cultivated with intensive agriculture -defined by the use of high amounts of manufactured nitrogen fertilizers- is consequently highly nitrogen polluted. Organic farming appears a promising approach to reduce N inputs in this basin as it excludes the use of synthetic fertilizers. The application and presence of N fertilizers might influence the activity, abundance and diversity of nitrifying and denitrifying bacterial communities. Therefore, the goal of this study was to compare the nitrifying and denitrifying 2 PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August community structure and diversity in soils subject to two different farming practices; conventional, with application of N fertilizer and organic, without the use of fertilizer. In addition we compared these community structures and diversity in the agricultural soils to those in adjacent forest and grassland soils. DNA was extracted from the different soil types and subsequently subjected to PCR-DGGE analysis. The whole bacterial community was studied by 16S rDNA analysis. The ammonia-oxidizing and denitrifying bacteria were targeted using the functional genes amoA (ammonia-monooxygenase) and nosZ (nitrous oxide reductase), respectively. We also enumerated the denitrifying community by the most probable number approach. Total bacterial species richness was similar for the different types of soil, whereas low similarity in 16 rDNA fingerprints indicated a high degree of heterogeneity among the different soil samples. The organic agricultural
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