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 soil, revealed a typical community composition for amoA and nosZ genes, indicating differences in nitrifying and denitrifying community structures compared to the other soils. Furthermore the nitrifying bacterial communities were significantly more diverse in organic soils (10–16 bands) compared to the other soils (5-10 bands). The denitrifying communities exhibited similar species richness (3-15 bands) and abundance (1.2-3.9 107 MPN/g dry soil) in the two agricultural practices. The identity of the ammonia-oxidizing bacterial community was further investigated by sequencing of amoA DGGE fragments from the DGGE gel. The amoA sequences were all affiliated with the genus Nitrosospira, commonly found in soil independent of the agricultural practices. The results of this study show that the bacterial community structure and richness are similar and seem not affected by the agricultural practices. Both nitrifying and denitrifying community structures differ between conventional and organic soils, moreover, a higher nitrifying richness was observed in organic, non-fertilized soils.

080B Effect of conversion of conventional pasture to intensive silvopastoral systems on soil bacterial communities Ziv Arbeli*1, Ana María Cubillos Cárdenas1, Victoria E. Vallejo1, Wilson Teran1, Richard P. Dick2, Fabio Roldan1 1Pontificia Universidad Javeriana, Colombia, 2The Ohio State University, Colombia

Conventional livestock pastures are one of the major causes of native-forest displacement and soil degradation in Latin America. Intensive silvopastoral systems, which integrate fodder shrubs planted at high densities (>10,000 plants ha−1), intercropped with highly-productive pastures and timber trees, has been developed in order to improve sustainability of livestock production and decrease its negative environmental impacts. These systems were first introduced to Colombia at “El Hatico” by a gradual conversion of conventional livestock pastures to intensive silvopastoral. Previous studies have shown that these systems increase the availability of forage and water during the extreme drought season, reduce the occurrence of pest and disease, and improve meat and milk production. Moreover, they improve soil quality, and increase the diversity of soil macroorganisms. In this study, we evaluated the effect of conversion of conventional pasture to intensive silvopastoral systems on soil bacterial communities. To attain this objective, we compared soil-bacterial-communities (PCR-DGGE of the gene 16S rRNA) of an intensive silvopastoral chronosequence (established 3-15 years ago) with an adjacent conventional pastures and a native forest. Additionally, we evaluated if the canopy of the main tree of these systems, Prosopis juliflora, affect these communities. Each treatment had three spatially separated replicates, and each replicate was comprised of two composite samples. The bacterial community structure of soils from intensive silvopastoral was more similar to that of the Forest than to the community of conventional pasture. Bacterial communities were principally influenced by ammonium, soil compaction and humidity. Additionally, community structures were more similar between oldest and intermediate silvopastoral systems than younger systems. On the other hand, the comparison of soil-bacterial-communities located under and outside tree's canopy (P. juliflora) yielded surprising results: although nutrient concentrations under the canopy was significantly higher, soil-bacterial-communities within the same paddock were similar independent of the presence of tree. This might indicate that geographical distance is an important factor for shaping soil-bacterial- communities. The main conclusion was that soil microbial communities of silvopastoral systems are more similar to those of native forest than to the comunity of conventional silvopastoral systems. It reaffirms that silvopastoral systems improve soil quality and biodiversity.

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PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August

081B The Effect of carbon input on the evolution of the soil microbial community in the central hyper-arid Namib Desert Alacia Armstrong1, Jean-Baptiste Ramond*1, Marla I. Tuffin1, Don A. Cowan2 1Institute for Microbial Biotechnlogy and Metagenomics (IMBM), University of the Western Cape, South Africa, 2Department of Genetics, University of Pretoria, South Africa

Characterized by high temperature fluctuations and hyper-aridity, the Namib Desert, considered the oldest desert in the world, is an extreme environment. In such an environment, an understanding of the relationship between the soil carbon cycle and the microbial community structure would aid in gaining a better understanding of the global carbon cycle, especially with regard to climate change. Indeed, seasonal grass represents a major carbon source in this hyper-arid ecozone and its impact on the soil microbial communities, the main drivers of ecosystem functions, have yet to be studied.

In order to do so, and to assess the reproducibility, variability and validity of our sampling strategy, a 8100 m2 plot covered with seasonal grass was divided into 81 10 x 10 m plots near the Gobabeb Research Station. Soil surface samples (0-3 cm depth) were collected on day 0, 3, 7, 15, 30, 45 and 60, from 8 randomly selected plots. Also, two adjacent 100 m2 control sites were selected on the observational absence of grass and sampled as above.

Terminal-Restriction Fragment Length Polymorphism (T-RFLP) was used to assess the evolution of the microbial communities over time. Soil microbial activity and biomass were measured using fluorescein diacetate (FDA) and ATP analysis, respectively. Soil temperature, moisture and chemical analysis were also monitored.

082B Diversity of methane-oxidizing bacteria in rice paddy field ecosystem: investigation by cultivation method and fluorescence in situ hybridization Susumu Asakawa*1, Dayéri Dianou2, Chihoko Ueno1, Takuya Ogiso1, Tran Van Huy1, Arata Katayama1, Makoto Kimura1 1Nagoya University, Japan, 2Centre National de la recherche Scientifique et technologique, Burkina Faso

Rice paddy field is one of the major sources of atmospheric methane and methane-oxidizing bacteria could play an important role in the mitigation of its emission. Based on phylogenetic, physiological, morphological and biochemical characteristics, methane-oxidizing bacteria are divided into two major subclasses: the Gamma- and Alpha-subclasses of Proteobacteria and designated as type I and type II methane-oxidizing bacteria, respectively. Molecular ecological studies provided accurate information on in situ diversity, community structure and abundance of methane-oxidizing bacteria in rice paddy field ecosystem. However, the physiological traits and the species composition of methane-oxidizing bacterial communities in this ecosystem are still not documented well. This study investigated diversity of cultivable methane-oxidizing bacteria in rice paddy field ecosystem by combined culture-dependent and fluorescence in situ hybridization techniques.

Seven microsites of a Japanese rice paddy field were focused in the study: floodwater, surface soil, bulk soil, rhizosphere soil, root, basal stem of rice plant, and rice stump of previous harvest. Based on pmoA genes analysis and transmission electron microscopic observation, four type I and nine type II methane-oxidizing bacterial isolates were obtained from the highest dilution series of enrichment cultures. In the type I methane-oxidizing bacteria, three isolates from floodwater and stump were closely related to Methylomonas spp.; one from rhizosphere soil was most related to Methyloccocus- Methylocaldum-Methylogaea clade. All the type II isolates were related to Methylocystis methanotrophs. Observation by fluorescence in situ hybridization confirmed the presence of both types I and II methane-oxidizing bacteria in all the microsites and in the related enrichment cultures as well.

The type I isolates included a novel species in the genus Methylomonas from floodwater, strain Fw12E-Y. Cells of strain Fw12E-Y were Gram-negative, motile rods with single polar flagellum and type I intracytoplasmic membrane arrangement. The strain grew only on methane or methanol as the sole carbon and energy source and optimal growth was achieved at 30ºC, pH 6.5 and with 0-0.1% (w/w) NaCl. Strain Fw12E-Y was most closely related to Methylomonas species based on the 4

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August sequence of 16S rRNA gene, but the similarity values were lower than 96%. Phylogenetic analysis of pmoA and mxaF genes indicated that the strain belonged to the genus Methylomonas (97 and 92% deduced amino acid sequence identities to M. methanica S1T, respectively). The DNA G+C content was 57.1 mol%. Chemotaxonomic data about major quinone (MQ-8) and major fatty acids (C16:1 and C14:0) also supported the affiliation of the strain to the genus Methylomonas. Based on the phenotypic, genomic and phylogenetic features, strain Fw12E-Y represented a new species, for which the name Methylomonas koyamae sp. nov. has been proposed. This is the first type I strain isolated from floodwater of a rice paddy field.

The study reported, for the first time, the diversity of cultivable methane-oxidizing bacteria including novel species in type I subclass in rice paddy field compartments. Refining growth media and culture conditions, in combination with molecular approaches, will allow to broaden our knowledge on methane-oxidizing bacterial community in rice paddy field ecosystem.

083B The microbial community structure of soil samples enriched with methanol at different pH and temperature Rozelin Aydin*1, Francien Peterse2, Stefan Schouten3, Jaap S Sinninghe Damsté3, Hauke Smidt4, Alfons JM Stams4 1Laboratory of Microbiology, Netherlands, 2ETH Zurich, Switserland, 3NIOZ, Netherlands, 4Wageningen University/Laboratory of Microbiology, Netherlands

Methylotrophic bacteria can use reduced one-carbon compounds, methane, methanol and methylated amines and other methylated compounds, as a sole of carbon and energy. They are playing an important role in global carbon cycle. Most of the methylotrophic bacteria are able to use methanol as a substrate. It was already shown that 80% of methylotrophic isolates were able to degrade methanol. All well-known methanol utilizing bacteria belong to Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia. In the present study we aimed to investigate environmental factors that could affect the composition of a methylotrophic enrichment culture. With a special interest, we also made an investigation on the branched GDGT lipids, used as a paleoenvironmental proxy for past soil pH and temperature, producers. To meet this goal, Têt watershed sample, Teso 2, was taken from southeast of France in 2007. Enrichment cultivation was perform under 6 different conditions with respect to pH and temperature. In order to characterize dynamics of microbial composition membrane PFLA profiles were measured and molecular analysis was done by barcoded pyrosequencing analysis of PCR-amplified 16S rRNA gene fragments. In total 6 phyla, Proteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, Acidobacteria, and several unclassified bacteria were detected. Besides the well-known methylotrophic genera we also found some novel methylotrophic genera including Pedobacter, Acidobacteria subdivision 1 and Janthibacterium . The results obtained using these assays will be presented and discussed further.

084B Microbial characterization of angled core samples of a zerovalent iron barrier in Belgium Rozelin Aydin*1, F Maphosa2, Q Simons3, H Sapion4, S van Roy3, N Hermans3, L Bastiaens3, H Smidt2 1Laboratory of Microbiology, Netherlands, 2Wageningen University/Laboratory of Microbiology, Netherlands, 3VITO, Belgium, 4SAPION, Belgium

A zerovalent iron (ZVI) barrier was implemented at a site contaminated with chlorinated aliphatic hydrocarbons in Belgium since 2005. The barrier is 200 m long and 30 cm wide, consisting of 20% fine ZVI and 80% of coarse sand, and it was installed on a clay layer situated between 3.5 and 6 m below the ground surface. The influence of this ZVI barrier on the microbial composition was analyzed in two angled cores, one within the contamination plume, and the other at the edge of the barrier outside the contamination plume. To get this insight barcoded pyrosequencing analysis of PCR-amplified 16S rRNA gene fragments was conducted. Microbial community composition was different between the two core samples, inside plume and outside the plume. The microbial compositions were also location- specific grouping separately depending on whether samples were taken from downstream, upstream, edge or inside the barrier. For example unclassified Desulfutomaculum which are gram positive sulfate reducers were the most abundant in the barrier whereas downstream was dominated by Acidobacteria 5

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August and Betaproteobacteria. Further analysis of barrier-derived samples showed that community composition consisted of Gram positive and negative sulfate reducing bacteria, iron reducing bacteria, methylotrophs and denitrifiers. Specifically, the vast majority of sequences belong to the Firmicutes, Proteobacteria, Tenericutes followed by TM6, Actinobacteria, Spirochetes, Chloroflexi, Acidobacteria, SPAM, NC10, Chlorobi and several unclassified. Our study showed the ZVI barrier influences the microbial community composition within the plume and appears that particular groups like, sulfate reducers and denitrifiers are higher inside the ZVI barrier. Detailed information on the core samples and bacterial community composition will be further discussed in the poster.

085B The mineral composition and presence of charcoal in artificial soils determine microbial community structure and the response to spiked phenanthrene Doreen Babin*1, Guo-Chun Ding2, Geertje Johanna Pronk3, Holger Heuer2, Katja Heister3, Ingrid Kögel-Knabner3, Kornelia Smalla2 1Julius-Kühn Institut, Germany, 2Julius-Kühn Institut, Federal Research Centre for Cultivated Plants, Germany, 3Technische Universität München, Lehrstuhl für Bodenkunde, Germany

In soil, organic, inorganic and biological constituents are contacting each other and forming large biogeochemical interfaces. To unravel soil-microbial interactions, this study aimed to get insights into how soil composition determines the establishment of microbial communities. Since the comparability of microbial communities from natural soils is problematic, 7 artificial soils varying in quartz sand, clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal were composed. Each artificial soil was inoculated with the same initial microbial community extracted from a natural Cambisol and autoclaved manure was used as nutrient source. Incubation was set up under constant environmental conditions (in the dark at 20°C). Sampling with subsequent extraction of total community DNA took place on days 1, 9, 31, 90 (short-term) and on day 360 and 450 (long-term), respectively. 16S rRNA gene and ITS amplicons for Bacteria or Fungi, respectively, were analyzed by denaturing gradient gel electrophoresis (DGGE). DGGE analysis revealed minerals and charcoal as shaping factors of microbial community composition. Pyrosequencing of day 90 samples confirmed a striking effect of charcoal on the bacterial community, and several discriminative taxa were identified between the artificial soils.

Furthermore, the response of established microbial communities at biogeochemical interfaces to persistent organic pollutants was explored. Therefore, one-year incubated artificial soils were spiked with phenanthrene as model compound (2 g/kg) and incubated for another 70 days. Cultivation and DGGE showed that the responses of Bacteria, Actinobacteria, Alphaproteobacteria and Betaproteobacteria to phenanthrene spiking differed depending on the artificial soil compositions and the taxonomic group analyzed. In contrast, a less pronounced effect of phenanthrene spiking was seen for Fungi. Cloning and sequencing of 16S rRNA gene of possible phenanthrene degrading isolates revealed mainly members of Actinobacteria and Firmicutes. PCR and hybridization showed an increased abundance of ring hydroxylating dioxygenase genes (PAH-RHDα) in response to phenanthrene spiking.

In conclusion, microbial communities in artificial soils shaped by minerals and charcoal responded differently to phenanthrene.

086B Hydraulically active biopores stimulate pesticide mineralization in agricultural subsoil Nora Badawi*1, Anders R. Johnsen1, Kristian K. Brandt2, Jan Sørensen2, Jens Aamand1 1GEUS, Denmark, 2University of Copenhagen, Dept. of Agriculture and Ecology, Denmark

Soil biopores can serve as preferential flow paths for downwards transport of inorganic nutrients and dissolved organic carbon. Pesticides may also be transported down through the subsoil in biopores, thereby posing a threat to the groundwater resource. On the other hand, biopores might constitute microbial high-activity hot spots for pesticide mineralization, thereby reducing the threat of pesticide leaching. To investigate this we identified hydraulically active biopores in a test plot of an agricultural field by percolating brilliant blue through the soil. Small portions of soil (200 mg) were then sampled at approx. 1-cm intervals along a transect across 10 biopores at two depths: 30 cm b.g.s. (transition zone below the plough layer) and 55 cm b.g.s (subsoil). The samples were used to determine heterotroph density (CFU), respiratory activity (soil respiration), growth activity ([3H]leucine incorporation), 6

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August bromoxynil and MCPA degrader density (most probable number), and bromoxynil and MCPA mineralization activity. Biopores did not increase heterotroph density, respiratory activity, growth activity, or bromoxynil mineralization in the transition zone, but slight increases were observed in the subsoil. In contrast, the density of MCPA degraders and MCPA mineralization activity were highly stimulated in the transition zone biopores. In the subsoil the density of MCPA degraders was significantly lower, and no MCPA mineralization occurred. We conclude that hydraulically active biopores may constitute high-activity hot spots for pesticide mineralization, but that this biopore effect is compound-specific and possibly inadequate to prevent pesticide leaching at increasing depths, where pesticide mineralization may be conducted by low-density degrader populations.

087B Exploring the structure and function of fungal communities in forest soils: challenges and limitations of current methodologies Petr Baldrian*, Tomáš Větrovský, Jana Voříšková, Ivana Eichlerová, Jaroslav Šnajdr, Lucia Žifčáková, Martina Štursová Institute of Microbiology ASCR, Czech Republic

The structure and function of soil fungal communities receives currently considerable attention. This is mainly due to the fact that fungi play a key role in the C and N cycling in soils, especially of the forest biomes. In addition, the recent establishment of high-throughput-sequencing methods, labelling with stable isotopes or metaproteomics offers a much higher resolution of the current studies. The interpretation of experimental results is, however, still challenging due to the fact that many methods may potentially contain more or less apparent biasses. This contribution aims to point at the most important limitations of current methodologies to explore fungal abundance, community composition and function in forest soils as studied using biomass quantification techniques (PLFA, ergosterol, qPCR), shotgun or amplicon-based next-generation-sequencing, stable isotope probing and environmental metaproteomics. In the case of microbial biomass surveys, our results show that various methods (rDNA quantification, PLFA or ergosterol assays) yield widely different results of the fungal biomass content or fungal/bacterial biomass ratio and identify the differences in the composition of fungal mycelia / genomes as a source of such errors. For the next-generation-sequencing data, we show that shotgun methods are not consistent with PCR-based methods and that the use of rDNA markers is highly biased due to uneven content of ITS copies per fungal genome as can be demonstrated if single-copy genes are sequenced. The approaches to explore the active fraction of the total fungal community by the analysis of RNA-derived sequencing or the use of Stable Isotope Probing and, most recently, environmental metaproteomics offer an attractive insight into the functioning of fungal communities. However, even the use of these methods must be careful in order to avoid experimental errors.

088B Microbial communities in ephemeral streams and the impact of saline mine discharge Janina Beyer-Robson*1, Philip Bond2, Sue Vink1 1Sustainable Minerals Institute, The University of Queensland, Australia, 2Advanced Water Management Centre, The University of Queensland, Australia

Ephemeral river systems are a major part of the Australian river landscape. In such systems major biogeochemical cycling occurs in the river sediments of the hyporheic zone where the surface and groundwater mix. Microorganisms play a key role in driving these biogeochemical reactions which are vital in maintaining the function and food webs of riverine ecosystems. In Australia, these river systems are increasingly subjected to salinization due to agriculture and industrial growth. In particular, concerns in regards to coal mining and saline mine discharge impacts on aquatic ecosystems have been highlighted in the Bowen Basin, Queensland. However, salinity impacts on biogeochemical processes and the microorganisms which drive these processes in ephemeral systems are poorly understood.

This study investigates the microbial communities in hyporheic sediments in the Isaac Catchment. The composition of microbial communities in the river sediments was examined using pyro-tag sequencing of the 16S ribosomal RNA gene. Shifts in microbial community composition occurred in relation to the variable environmental conditions in the ephemeral river system, in particular in relation to sediment moisture, pH and the availability of carbon. The effects of saline mine water on microbial community composition upstream and downstream from mine discharge points were also examined. Laboratory 7

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August incubations were conducted to examine the effect of salinity on major river ecosystem process rates. This revealed that that process rates were carbon limited in these ephemeral systems, while salinities over 5000 µS/cm were required to have an influence on process rates.

The influence of salinity on nutrient dynamics and microbial mediated ecosystem processes will provide insights for managing such ephemeral rivers. The outcomes will support environmental management positions, in particular towards the implications of saline discharge from coal in the region.

089B Characteristics of metal-tolerant bacteria and their plasmids isolated from a platinum mine tailings dam in South Africa Carlos Bezuidenhout*, Tladi Mahlatsi, Laurette Marais, Mark Maboeta North-West University: Potchefstroom Campus, South Africa

Mining in South Africa annually contributes over US$7 billion to the country's gross domestic product. This, however, comes at a significant environmental cost which include water, air and soil pollution as well as the generation of domestic and hazardous wastes. Metal contamination from mining may thus have profound effects on microorganisms in the environment. High concentrations of metals in soil and aquatic environments are evolutionary pressures selecting for microorganisms tolerant to these metals. Metal tolerance maybe conferred to these organisms by mobile genetic elements such as plasmids. In this study, soil samples collected on and in the vincinity of a platinum mine tailings dam were analysed to determine the diversity and physiological characteristics of metal tolerant bacterial strains. Bacteria were isolated and purified on 0.1% nutrient agar containing a combination of aluminium and nickel (8mM). Isolates were differentiated using morphological features and biochemical tests. Identification by sequencing of PCR amplified 16S rDNA fragments indicated Paenibacillus lautus, Stenotrophomonas maltophilia, Bacillus cereus, Bacillus thuringiensis, Paenibacillus validus, Paenibacillus ginsengagri and Alcaligenes faecalis as dominant species. Some strains were able to grow at temperatures ranging from 5°C to 45°C. Metal MIC's of the species were determined for a selection of metals and some were greater than 5 mM. Growth rates in the presence of these metals showed effects of inhibition as well as stimulation. The biosorption potential of the bacterial species was determined using transmission electron microscopy and electron dispersive x- ray. These methods indicated that some of the strains adsorbed up to 5mM of certain metals. Plasmids associated with the metal tolerance were isolated and transformed into E. coli JM109 rendering the latter strain also metal tolerant. Transformed E. coli JM109 were resistant to high concentrations of Ni2+/Al3+, Pb2+ and Ba2+, and were able to grow at concentrations of 10 mM or more of these metals. However, all the transformants were susceptible to Co2+ and Hg2+. The order of metal resistance was Ni/Al=Pb>Ba>Mn>Cr>Cu>Co=Hg. Plasmids were also re-isolated from the transformants and these plasmids were of the same size as the original ones. In order to determine the compatibility group, plasmids were subjected to PCR amplification using IncQ, IncP and IncW specific primers. Positive IncW PCR results were obtained for three of the plasmids. These were originally isolated from Paenibacillus ginsingari, Paenibacillus lautus and Bacillus cereus. To demonstrate that the plasmids were free of genomic DNA, a 16S rDNA PCR test was included. Plasmids that were positive for IncW PCRs were all negative for the rDNA PCRs. All plasmids in this study were also stably inherited, a feature associated with IncW plasmids. Results from this study demonstrated the potential ecological role of certain metal tolerant bacterial species as well as plasmids in heavy metal contaminated soil. Growth characteristics of these species suggest that they could possibly be exploited in remediation applications. Furthermore, more detailed genetic characterization of the plasmids is required. Plasmids isolated and characterised in this study may also hold biotechnology possibilities.

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PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August

090B Impact of nutrient addition on microbial community decomposition of wheat residues and carbon sequestration assessed with 13C labelled wheat Andrew Bissett*1, Clive Kirkby1, Maria Luisa Gutierrez Zamora2, Mark Brwon2, Peter Thrall1, Richardson Alan1 1CSIRO Plant Industry, Australia, 2School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia

Soils represent a significant terrestrial carbon reservoir and there is considerable interest in increasing or maintaining its size. Recent work has suggested that microbially mediated carbon sequestration into soils may be maximised by ensuring adequate supply of other nutrients (nitrogen, phosphorous, sulphur), thus maintaining high microbial carbon use efficiency. This is particularly important in cropping agriculture systems where appropriate crop-residue management may result in substantial soil carbon and general soil quality improvements.

A soil microcosm experiment was conducted to evaluate the effects of nutrients on the decomposition of wheat residues; specifically the effects on active bacterial community and carbon sequestration. Stable Isotope Probing (SIP) of 16S rDNA using Phylochip microarrays was employed to investigate the bacterial community associated with the decomposition of 13C-labelled wheat straw and incorporation of the carbon into soil humus. Respiration and carbon, nitrogen, phosphorous and sulphur transformations were measured over a 56 day incubation to assess priming effects, both gross and net humification efficiency and carbon sequestration through microbiological action. Soils that received wheat residues only always showed an increase in “new” carbon (13C), although they did not always show an increase in “total” (12C+13C) carbon. Soils receiving nutrients (nitrogen, phosphorous and sulphur) with wheat residues did show an increase in both new and total carbon pools, suggesting availability of nutrients other than carbon influence soil carbon sequestration. Bacterial communities responsible for residue decomposition and carbon sequestration were different for nutrient added and no-nutrient treatments, indicating microbial carbon use efficiency is also influenced by the composition of the active community. Our results suggest that soil carbon:nitrogen:phosphorus:sulphur (C:N:P:S) stoichiometry and bacterial community composition play important roles in determining potential levels of carbon sequestration in agricultural soils.

091B Carbon isotope fractionation of Italian rice field soil under H2/CO2 and different temperature regimes Martin Blaser*, Ralf Conrad Max-Planck-Institute for Terrestrial Microbiology, Germany

In anoxic environments organic matter is fermented to short chain fatty acids, alcohols as well as CO2 and H2. The two gaseous products can be further converted to either methane by methanogenic archaea or to acetate by acetogenic bacteria. Methanogenesis is energetically more favourable than acetogenesis. Nevertheless acetogens can outcompete methanogens at low temperatures. To investigate the contribution of both processes we incubated anoxic rice slurry under H2/CO2 at 15°C, 30°C and 50°C and followed the isotopic signatures of the carbon compounds (CO2, CH4, acetate) by mass spectrometry. For better differentiation of the two processes a second incubation was performed with bromoethanesulfonate an inhibitor of methanogenesis.

092B Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding* Aarhus University, Dept. of Environmental Science, Denmark

Soils provide numerous essential ecosystem services such as carbon cycling, recycling of nutrients and waste, soil remediation, plant growth support and regulation of above ground biodiversity, resilience, and soil suppressiveness. As such, soil ecosystem services are beneficial and vital for human life and at the same time threatened by anthropogenic activities. Increasing awareness of the threats to soil and the ecosystem services has fostered the need for a thorough understanding of soil functions and activities. Soils are very species-rich and show great functional heterogeneity. While molecular analysis of gene expression is developing, simple and inexpensive indicators of soil microbial functions is still needed. 9

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August

In soil, enzymes originate from a variety of organisms, notably fungi and bacteria and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity will reflect the functional diversity and activity of the microorganisms involved in decomposition processes. Their activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysacharides as cellulose, hemicellulose and chitin, while degradation of proteins has been followed by amino-methyl-coumaric substrates (AMC).

Based on these fluorogenic substrates the Extracellular Enzyme Activity assay was optimized as a microwell based standardized assay for the activity of enzymes involved in degradation of polysaccharides and proteins. Using specific MUF and AMC substrates on soils from across Europe and based on literature data the experimental conditions of extraction of enzymes from soils, use of buffer and pH, substrate concentration, temperature and the necessary controls to include were optimized and standardized. This has resulted in an optimized standard operating procedure of the assay which will be tested in a soil transect across Europe as an indicator of soil functions.

093B Degradation of fungal mycelia in soil: decomposition rate and microbial community analysis Vendula Brabcova*, Monika Nováková, Anna Davidová, Petr Baldrian Institute of Microbiology, ASCR, Czech Republic

Fungal mycelia represent the major part of the microbial biomass in forest soils. It originates mainly from saprotrophic fungi degrading plant litter and ectomycorhizal fungi dominating deeper soil horizons. The most important components of fungal cell walls are polysaccharides counting 80-90% of the total. As fungal cell walls contain chitin and protein in relatively high concentrations, their tissues are thus significant pools of carbon and nitrogen. However, neither degradation rate and factors affecting decomposition nor microbial community involved are well understood yet. The aim of this study was to determinate the physiological and chemicals changes during mycelia degradation measured as loss of dry matter and activities of extracellular enzymes (α- and β-glucosidase, cellobiohydrolase, β-xylosidase, β-mannosidase, α-galactosidase, chitinase, acid phosphatase and aminopeptidases). The biomass and microbial community structure was characterized using general bacterial and fungal TRFLP and qPCR. Mycobags with two types of freeze dried mycelia (Rusulla spp. and Tylopilus felleus) placed between litter and humus soil horizons were sampled each second week for 8 weeks, surrounding soil was used as control. Study site was located in Xaverov forest, Czech Republic. Very fast degradation within 2 weeks (57% of dry mass) slowed towards the end of the experiment - 67% respective 85% of dry mass depending on mycelia type. Measured enzyme activities differed significantly between soil and degraded mycelia as well as between two mycelia types. Generally, enzyme activities in degraded mycelia were higher. No significant peak activities were detected over the experiment. Relative enzyme activity patterns showed differences among treatments: mycelia were dominated by phosphatase and peptidases (approx. 40% each), whereas soil solely by phosphatase (up 80%). qPCR pointed to significantly higher bacterial biomass in degraded mycelium with maximum in 6 week, whereas fungal biomass was rather constant. TRFLP proved changes in bacterial community in degraded mycelium compared to soil. Differences between the mycelia types and sampling dates were less detectable. Fungal community showed no significant variation between the treatments or in time. Principal component analysis of all results showed clear differences between soil control and mycobags. Changes between mycobags during decomposition were also detected but these were not always significant. Statistical analysis did not proved the clear differences in degradation between the mycelia types. Our results show that fungal mycelium degradation is very fast compared to plant litter. Range of produced extracellular enzymes during mycelia degradation reflects the main chemical composition of mycelia and differs from soil extracellular enzyme pattern. The mycelia degradation is due to unchanged fungal community mainly driven by bacteria. Detailed study of microbial community structure and function using molecular approach will follow this experiment to describe the details of microbial community composition and function.

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094B Functional diversity and quantification of catabolic genes (BPH) in "Amazonian dark earth" and its adjacent soils Maria Brossi*1, Mariana Germano1, Lucas Mendes1, Shoko Iwai2, Siu Tsai1 1University of São Paulo - Center of Nuclear Energy in Agriculture, Brazil 2University of California, USA

Soils located in the Amazon region are generelly regarded as unsuitable for agriculture practices due to their low content of organic matter and low pH. However, this region also presents extremely fertility soils, the Amazon Dark Earth (ADE). These Anthrosols contain thick, dark-colored surface horizons, approximately three times more organic matter than the original soil, nitrogen and phosphorus and the presence of both pottery sherds besides it charred biomass of unknown organic origin - biochar. The original soils from Amazon, called as adjacent soils (ADJ) have no history of anthropogenic activities. Here we present a study using soils from ADE and ADJ sites under different land uses, (agriculture and secondary forest) regarding the diversity and quantity of the catabolic gene (bph) and directly cross comparing. Culture-independent approaches, such as Pyrosequencing (454 Life Sciences, GS FLX Titanium) was conducted to assess the diversity and qPCR to quantify the catabolic gene (bph) encoding for the catalytic subunit of aromatic dioxygenases, responsible by the degradation of aromatic hydrocarbons. The pyrosequencing generated a total of 5,959 valid sequences, yieldind a total of 862 and 751 sequences for ADE soil under secondary forest and under agriculture and 1919 and 2427 sequences for ADJ soil under secondary forest and under agriculture, respectively. A total of 111, 91, 152, and 237 Operational Protein Families (OPFs) (cut-off value of 0.06, MOTHUR) were observed for ADE under forest and under agriculture and ADJ under forest and under agriculture, respectively. Jackknife, Chao1 and ACE richness indices were determined, as well as Shannon- Weaver and Simpson diversity indices. Data from bphgene pyrosequence indicated that this gene diversity was higher in ADE soils than in ADJ soils (significance <0.0001, LibShuff), maybe suggesting that the anthropogenic action might influence in this gene diversity. The qPCR results showed that ADE soils (under forest and under agriculture) presented a higher number of bphgene copy numbers (1.4 x 106 copies soil gram -1 for TPA under forest and 9 x 105 copies soil gram -1 l for TPA under agriculture) than its adjacent soils (under forest and agriculture) (1.3 x 105 copies soil gram -1 for ADJ under forest and 5.2 x 105 copies soil gram -1 for ADJ under agriculture). These results supply new insights into the distribution of the diversity and the quantity of this catabolic gene in anthropic soils and highlight the need for further studies on factors that could handle functional diversity in Amazonian soils.

095B Enzyme kinetics and temperature sensitivity in Arctic permafrost soils Jiří Bárta*1, Markéta Applová1, Iva Lacmanová1, Petr Čapek1, Kateřina Diáková1, Hana Šantrůčková1, Andreas Richter2 1Department of Ecosystem Biology, University of South Bohemia, Czech Republic, 2Department Terrestrial Ecosystem Research, University of Vienna, Austria

Soil enzymes play crucial role in transformation of organic matter and are key players in biogeochemical cycling of nutrients. Arctic soils are one of the most sensitive ecosystems to global changes. Global climatic models predict in Arctic higher increase of temperature in the following decades compared to other terrestrial ecosystems. Most of the changes are predicted to take place in the organic top soil horizons. However, significant portion of organic material in Arctic is buried in deeper soil profile by cryoturbations. Very little is known about temperature sensitivity and kinetics of soil enzymes in these cryoturbations.

The goal of our study was to determine the Q10 values of maximum enzymatic rate (Vmax) and enzyme substrate affinity (Km) of selected soil enzymes (phosphatase, β-glucosidase, cellobiosidase, alanine-aminopeptidase and chitinase) and compare these values in top soil and cryoturbations.

Potential enzyme activity was determined by standard fluorometric method using 4- methylumbelliferone and 7-amido-4-methylcoumarin. Enzyme kinetic parameters (Km, Vmax) in the temperature interval from 0 to 40°C were determined.

We found one to two orders lower Vmax in cryoturbations than in top soil. Km values almost did not change indicating that the isoenzyme pool showed similar substrate affinity with increasing 11

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August

temperature. In cryoturbations we found high temperature sensitivity of β-glucosidase Vmax (Q10 = 2.5) which was similar to the top soil (Q10 = 2.4) indicating similar response to increasing temperature and potential organic carbon mineralization. On the other hand, temperature sensitivity of peptidases and phosphatases was lower in cryoturbations. This can lead to misbalance in organic carbon, nitrogen and phosphorus mineralization because carbon will be mineralized at higher temperature faster and can become the limiting step in organic matter decomposition.

096B Factors influencing the bacterial community structure and distribution in northern peatlands Hinsby Cadillo-Quiroz*1, Stephen Zinder2, Joseph Yavitt2 1Arizona State University, USA, 2Cornell University, USA

Northern peatlands store nearly a third of all terrestrial carbon and have an important role in the global C budget and methane emissions. These ecosystems have a broad distribution including northern regions with different climates, and also present a diverse range of ecological conditions.

Although a few studies have identified Bacteria in peatlands, their community composition has only been evaluated in a narrow geographical scale, and the effects of climate and ecological variables on their bacterial communities have not been evaluated simultaneously. We hypothesize that climate and peatland type can differentially structure bacterial communities.

In this study we sampled 22 peatlands from Northern America and Europe encompassing the two climatic regions were peatlands are most abundant (temperate and boreal), as well as an oligotrophic to minerotrophic ecological gradient (bog to fen). We recorded multiple environmental variables including climate, pH, vegetation, and peatland classification. We evaluated the bacterial community composition by assessing the 16S rRNA gene composition by 454 pyrosequencing of a 500 bp fragments containing the V4 and V5 region. An average of 7000 reads with high quality scores were retrieved and used for analysis with Qiime software.

Potential respiration assays showed all sites as capable of methane and CO2 production with variable rates. 454 pyrosequencing of the 16S gene demonstrated a highly variable composition among bacteria in all sites. Diversity of observed OTUs at 97% similarity ranged from around 1000 to over 5000 groups; although diversity estimators suggest values nearly 5 times higher. pH followed by ecosystem type were strong predictors of the overall diversity with higher values at near neutral pH, while diversity levels by climate region were not statistically differentiable. Complementary community structure and bacterial classification analysis demonstrate that pH and ecosystem type were the main factors structuring bacterial composition and that climate also had significant effects. For instance, when considering the differential abundance of dominant bacterial groups among acidic sites, we determined that Acidobacteria Gp1, Syntrophobacteria, Verrucomicrobia have a higher representation in acidic temperate sites, while Dehalococcoidetes and Acidobacteria Gp 2 were more abundant in acidic boreal sites. Other, mostly heterotrophic, groups such as beta proteobacteria, bacteroidetes, and actinobacteria were better represented in minerotrophic fens. Statistical correlation analysis is also providing evidence for pH as well as temperature regulation in the abundance of classified bacterial groups.

Our initial report presents evidence of ecological and climate influence on the bacterial community composition in peatlands spanning a broad range of ecological and climate conditions from northern latitudes.

097B Effects of plants materials on the dissipation of herbicide pendimethalin in soil and its microbial communities Bing-Sheng Chen*, Fang-Yu Hsu, Jui-Hung Yen Department of Agricultural Chemistry, National Taiwan University, Taiwan

Green manure is primarily used in environmentally friendly agricultural practices to reduce the application of chemical fertilizer for many years. However, the effect of green manure on dissipation of herbicide and soil bacteria community was rarely discussed. In this study, we tried to discuss the effect

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PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August of lupin (Lupinus luteus) on the dissipation of pendimethalin in Sankengtzu (pH 5.2) and Erhlin (pH 7.7) series soil by high performance liquid chromatography (HPLC), and the soil bacterial community was also studied by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) during the dissipation period. According to the results, the half-life of pendimethalin was 34.8 and 62.2 days in Erhlin and Sankengtzu soil, and became 46.3 and 49 days after amending lupin, respectively. The lupin amended would be beneficial to degrade of pendimethalin in Sankengtzu soil. The PCR- DGGE results show that the microbial communities of blank treatment were relatively close with the green manure treatment, but separated after applied pendimethalin in 110 days. Moreover, compare to Sankengtzu soil, the microbial communities of lupin treatment was not so influenced by pendimethalin in Erhlin soil. The study indicates that green manure and soil pH could affect the dissipation of pendimethalin and green manure would protect soil microbial communities from pendimethalin.

098B Changes in soil microbial community structure and metabolic activity following the convertion from the native Pinus massoniana plantations to the exotic Eucalyptus urophylla × grandis plantations Falin Chen*, Kai Zhang, Yifeng Li, Hua Zheng State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, China

Deforestation and afforestation with fast growing exotic species are the key land-use changes throughout China and even the world. However, there have been few studies on the effects of exotic species planting on soil microbial communities which mediate nearly all biogeochemical processes in terrestrial ecosystems. To understand the impacts of extensively exotic species planting on the structure and function of soil microbial communities, we made comparison of the biomass, the phospholipid fatty acid (PLFA) composition and metabolic activities (BIOLOG profiles) of soil microbial communities under 9 pairs of Eucalyptus urophylla × grandis plantations and adjacent native Pinus massoniana plantations in the subtropical forest areas of Guangxi province, south China. The results showed that: the biomass, the abundances of each microbial PLFA (as indicated by characteristic PLFA of gram+, gram- bacteria, AM fungi, fungi, actinomycetes) and the total PLFA of soil microbial communities in exotic Eucalyptus urophylla × grandis plantations were significantly lower than those of former native Pinus massoniana plantations. The ratios of monounsaturated to saturated fatty acid, gram+ to gram- bacteria, iso- to anteiso- branched PLFA and cy19:0 to 18:1ω7c which reflect the soil environmental and nutritious stress, were significantly higher in the eucalyptus plantations soils. The metabolic activity, richness and diversity of soil microbial community (as indicated by soil BIOLOG profiles) in the eucalyptus plantations soils were significantly lower than these of adjacent pine plantations. The changes of soil microbial community structure and metabolic activity were partly explained by the decreased soil moisture, total C, total N, soil organic carbon and available N, following the convertion from the native Pinus massoniana plantations to the exotic eucalyptus plantations. The studies suggested that the convertion from the native pine plantations to the exotic eucalyptus plantations not only increased the soil environmental and nutritious stress, but also decreased diversity and metabolic activity of soil microbial communities.

099B Plasmid isolation and characterization from a biofilter system used for pesticide removal María Del Papa*1, María Martini2, María Salas2, Jose López2, Ileana Salto2, María Giusti2, Mauricio Lozano2, Gonzalo Torres Tejerizo2, Mariano Pistorio2, Andreas Schlüter3, Alfred Pühler3, Antonio Lagares2 1 Universidad Nacional de La Plata-UNLP, Argentina, 2Instituto de Biotecnología y Biología Molecular-IBBM-CONICET-UNLP, Argentina, 3Lehrstuhl für Genetik, Universität Bielefeld, Germany

A systematic search of genetic markers of interest, was performed over different terrestrial and aquatic habitats towards their deeper metagenomic analysis. Based on the result of such genetic pre- screening, a biofilter system implemented for decontamination of pesticides used in agriculture was selected for further studies. The target biofilter consists of a biological active matrix that retains/degrades pesticides into its organic matter.

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In order to investigate the type and diversity of the information encoded in the plasmid mobilome present in the biofilter, a plasmid search over more than 1,400 randomly selected bacterial clones was performed using in situ-lysis gel electrophoresis (Eckhardt protocol). The experimental approach allowed for the identification of 75 plasmid-containing clones, with molecular weights ranging from 30 to 300 Kpb. Interestingly, almost 50% of the identified plasmids presented more than 100 Kbp in length. According to the plasmid profiles observed, the Isolates could be classified into 35 different diversity groups. From them, a sub-collection of clones representing all observed plasmid profiles were analyzed for the presence of replication functions present in the broad host range and conjugative IncP, IncN, IncW plasmids, and in the mobilizable IncQ plasmids. Over the purified collection of plasmids, a PCR-based screening was performed to identify clones carrying genes encoding for specific aliphatic and aromatic halogenases and dehalogenases, and for other enzymes of industrial interest such as laccases. Several plasmids resulted positive for these assays.

Based on the observed diversity, the high molecular weight of most plasmids, and the marker genes that we found on them, we approached the deep sequencing of our plasmid collection by using the Ion Torrent technology. The total non-redundant DNA sequence accounted for more than 7 Mb, and has been automatically annotated via the GenDB pipeline available at the CeBiTec (Uni-Bielefeld, Germany). Genes of interest, as identified from the sequencing and bioinformatics analysis, are physically available in the conserved plasmid collection and can be recovered for activity evaluation. The quite diverse replication/mobilization elements that were identified and sequenced also provide a diverse set of functional modules that can be useful for the design of new (cloning/expression) plasmid vectors suitable for their evaluation and use in non-conventional environmental bacterial hosts.

100B Influence of management practices on microbial consortia in paddy soils Thilo Eickhorst*, Hannes Schmidt, Stefan Knauth, Rolf Tippkötter University of Bremen, Soil Science, Germany

The cultivation of rice is of great importance for the feeding of the earth’s population. The management of paddy soils used for lowland rice cultivation is an important factor indirectly influencing the emission rates of greenhouse gases (CH4, CO2, N2O) and practices like puddling and seasonal irrigation of the soils mainly influence the physicochemical conditions in the soil matrix. After flooding the oxygen in the submerged bulk soil is rapidly depleted while the soil-water interface and the rhizospheric soil in the vicinity of active roots form microenvironments with different oxygen levels. At maturity of the rice plant, the commonly practiced termination of the irrigation of rice paddies again changes the physicochemical conditions in these soils.

To study the impact of these management-induced factors on the fate of microbial communities three Chinese paddy soils of different texture were used to conduct microcosm experiments under simulated climatic conditions. The soils were managed as described above and values of pH, redox potential, volumetric water content, and matric potential were collected continuously. In addition, the development of redox areas in the root-zone was regularly monitored via digital image analysis. Quantitative and qualitative analyses of microbial communities in the root-zone were conducted by the application of rRNA targeted catalyzed reporter deposition fluorescence in situ hybridization (CARD- FISH) and fingerprinting techniques (PCR-DGGE) combined with sequencing. Particular focus was drawn towards the rhizosphere, where interactions of soil and roots establish an unique and ever- changing environment for microorganisms.

Among the management-induced factors influencing the physicochemical environment in paddy soils, especially the terminated irrigation at maturity of the rice plant had a significant impact on the community structure of microbes. The relative abundance of CARD-FISH enumerated archaea and bacteria consistently decreased after drainage indicating a reduction in their metabolic activity. Furthermore, DGGE-fingerprints confirmed shifts in the composition of archaeal and bacterial communities particularly for the bulk soil samples. However, the metabolic activity of the plant also seems to considerably alter the occurrence and composition of microbial consortia in paddy soils. While the absolute abundance of archaea in the root-free bulk soil remained on a level of approximately 3.5×108 cells per gram of soil during the irrigated period, a continuous increase of archaeal cell numbers was found in the rhizospheric soil (up to 5.4×108). At the same time, the relative abundance of methanogenic archaea detected with the probes EURY499 and MSMX860 was found to 14

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August decrease in rhizosphere, indicating less favourable conditions for methanogens. This effect was particularly pronounced during the vegetative stage of rice plant development (tillering), which correlated with high rates of radial oxygen loss via roots observed by digital image analyses.

Further insights into the influence of both, human and plant-deduced effects on the spatiotemporal distribution of microbes in paddy soils are gained in ongoing experiments which are essential for an understanding of soil-microbe-plant interactions in highly dynamic microenvironments such as the rhizosphere of lowland rice.

101B Microbial diversity and physicochemical properties of soil in the lead (Pb) and zinc (Zn) mining area of Ishiagu, Nigeria Uchechi Ekwenye*, Chiedozie Nwachukwu Michael Okpara University of Agriculture,Umudike, Nigeria

In metal mining areas like Ishiagu, Ebonyi State in Nigeria, pollution by heavy metals is very significant but reduces with increasing distance away from mining sites. The effect of Zn and Pb contamination due to mining activities on the microbial diversity and physicochemical properties of soil in the Pb and Zn mining area of Ishiagu, Ebonyi state, Nigeria were investigated. Soil samples were collected from six sampling points; the mining pits, the waste dumps, the centre of the distance between the mining pits and the waste dumps,100m and 200m away from the mining pits and a distance far away from the sites(1.0km) as the control. The experiment was replicated in three mining sites. The extent of Pb and Zn contamination was determined using atomic absorption spectrophotometer and the results revealed that the area was highly contaminated by Pb and Zn with the extent of contamination decreasing as the distance from the mining site increases. The highest concentrations of Pb and Zn were found at the mining pit (source of contamination) Pb-263,008mg/kg and Zn-210,676mg/kg, while the least concentrations were at the control site (Pb-233mg/kg and Zn-283mg/kg).The results of the physicochemical analyses showed that the pH and the concentration of all the other chemical properties investigated were lowest at the mining pits followed by the waste dumps but increased as the distance from mining sites increases, corresponding with a decrease in the concentration of all the heavy metals. All microbial parameters analyzed were adversely affected by increasing concentrations of Pb and Zn with the least microbial loads found at the mining pits where concentrations of the metals were highest. The counts of total heterotrophic bacteria, coliform bacteria, spore-forming bacteria, oligotrophic bacteria and fungi were 2.987x103fu/g,9.88x101cfu/g,3.713x102cfu/g,3.22x102cfu/g and 5.03x102cfu/g at the pits and 1.91x107cfu/g,4.089x104cfu/g,9.20x105cfu/g,9.196x103cfu/g and 3.50x105cfu/g at the control respectively. A total of seven species of bacteria; Bacillus, Pseudomonas, Staphylococcus aureus, Micrococcus, E.coli, Azotobacter and Proteus, as well as six species of fungi;Fusarium, Penicillium, Aspergillus, Candida, Rhizopus and Mucor were isolated in the study. Species of Bacillus, Pseudomonas, Fusarium and Aspergillus were found to tolerate high levels of Pb and Zn concentrations. This study therefore shows that contamination by Pb and Zn due to mining activities adversely affected both the microbial diversity and the physicochemical properties of Ishiagu mining area.

102B Changes of soil microbiome due to repeated addition of manure from oxytetracycline- treated dairy cow Dana Elhottova*1, Martina Kyselkova1, Robert Bradley2, Heike Schmitt3, Jiri Jirout1, Nadezda Vrchotova4, Nancy Allard2, Havlickova Petra1 1Biology Centre ASCR, Institute of Soil Biology, Czech Republic, 2Sherbrooke University, Faculty of Science, Canada, 3Utrecht University, Institute for Risk Assessment Sciences, Netherlands, 4Global Change Research Centre ASCR, Lab Metabol & Isotop Anal, Czech Republic

The intensification of livestock production is typically accompanied by antibiotics application that is suspected to affect a microbiome of manures and manured soils.

We report on a microcosm experiment in which we monitored microbiome of soils amended with oxytetracycline-treated (OE) and non-treated (E) dairy cow manure, as well as non-amended (control) soils. The soils were collected from three farms that had not been fertilized with manure for at least 20 years. The treatments were applied to each soil (n=4) and these were incubated for two months. We 15

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August analysed (i) total microbiome (EL/NEL PLFA), (ii) culturable microbiome (total and tetracycline resistant (TET-r) bacterial counts on TSBA -/+ 0.003% TET; isolate identification 16SrRNA sequencing) and (iii) tetracycline resistome (PCR of otr(B), tet(B), tet(L), tet(M), tet(V), tet(O) and qPCR of tet(W), tet(Q), tet(Z).

We found (i) that PLFA profiles of soil microbiome were significantly affected by addition of both types of manure and PLFA profiles of OE-treated soils were specifically enriched with branched fatty acids and their hydroxy-derivates typical for Gram-positive bacteria and Bacteroidetes, respectively. (ii) Prevalence of culturable TET-r bacteria increased both in OE and E amended soils from 4% to 11% and 7%, respectively. The TET-r bacteria isolated from both of OE- and E-soils were enriched with Arthrobacter, Bacteroidetes, Flavobacterium, and Stenotrophomonas. Escherichia, Leucobacter, Streptomyces and Pseudomonas were isolated only from OE-soils. (iii) The OE and E manures did not differ in the total number of TET-r genes detected, and both contained tet(L), tet(M), tet(Z), tet(Q), tet(W) genes out of which tet(Z), tet(M), tet(W) were transferred to the soils in a reproducible manner.

We can summarise that cow manure were a source of TET-r genes as well as TET-r culturable bacteria that were transferred to soils, regardless of whether cows were actively undergoing tetracycline treatment. Nevertheless, differences between soils amended with TET-treated and non- treated manure were also found in total as well as culturable microbiome.

103B Transport of BAM degrading bacteria facilitated by fungal hyphae increases BAM mineralization in sand Berith Elkær Knudsen*1, Lea Ellegaard-Jensen2, Christian Nyrop Albers1, Søren Rosendahl2, Jens Aamand1 1GEUS, Denmark, 2University of Copenhagen, Denmark

Leaching of pesticides and their metabolites pose a great threat to the groundwater resources. The most frequently detected pollutant in Danish groundwater wells is the pesticide metabolite 2,6- dichlorobenzamide (BAM), which originates from the herbicide dichlobenil. Bioaugmentation has been suggested as a possible means to clean pesticide contaminated sites, as several strains capable of degrading for example BAM have been identified. There are however some challenges to overcome for bioremediation to be successful. It is difficult to ensure survival of introduced strains and studies have shown that bacteria applied to a surface rarely are transported below 5 cm into the soil, without the aid of some transport agent like for example fungi. Furthermore, the contaminants are often only present at low concentrations and are not easily accessible for the degrader bacteria.

The aim of this study was to test whether presence of the zygomycete Mortierella sp. LEJ702 affected BAM mineralization by Aminobacter sp. strain MSH1 in sand with different moisture contents. The hypothesis was that fungal growth facilitates transport of bacteria, and thus increases the bioavailability of BAM, leading to an increased mineralization.

14 14 Mineralization was determined using C-labeled BAM and measuring production of CO2. Furthermore, Thin Layer Chromatography (TLC) was performed to determine the fate of the BAM that had not been mineralized. Transport of Aminobacter sp. strain MSH1 was detected and quantified by PCR and QPCR respectively.

Results showed an increased mineralization rate by the Aminobacter-Mortierella consortia at all moisture contents. Moreover an overall greater mineralization was obtained by the consortia at the lower moisture contents. TLC results support these findings. Preliminary results of the QPCR indicate there had been a greater transport of Aminobacter through the sand columns when Mortierella was present.

The results indicate that fungal-bacterial consortia may be a promising strategy for obtaining successful bioremediation of pesticide contaminated sites.

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104B Degradation of the herbicide diuron by different Mortierella strains Lea Ellegaard-Jensen*1, Jens Aamand2, Søren Rosendahl1 1University of Copenhagen, Denmark, 2Geological Survey of Denmark and Greenland (GEUS), Denmark

Microbial pesticide degradation studies have until now mainly focused on the role of bacteria, although fungi have also been shown to degrade pesticides. In this study we clarify the background for the ability of the common soil fungus Mortierella to degrade the phenylurea herbicide diuron. Diuron degradation potentials of five Mortierella strains were compared, and the role of carbon and nitrogen for the degradation process was investigated.

Fungal strains were isolated from a soil previously treated with phenylurea herbicide. The isolates were identified after DNA extraction, PCR and sequencing, and five Mortierella strains were selected for further investigations. Degradation experiments were conducted to investigate the degradation potentials of the five strains. Additionally, the effect of different carbon and nitrogen levels on the degradation process was studied.

It was shown that strains able to degrade diuron constituted a closely related group, indicating that diuron degradation was linked to the phylogenetic position of the strains. Degradation of diuron was fastest in carbon and nitrogen rich media while deficiency of these constituents resulted in limited degradation, demonstrating that Mortierella do not utilize diuron as carbon or nitrogen sources. Diuron degradation was followed by formation of 1-(3,4-dichlorophenyl)-3-methylurea, 1-(3,4- dichlorophenyl)urea and a not yet identified metabolite.

In conclusion, this work underlines the relevance of including fungi in future research on the fate of diuron in soil including its degradation pathways.

105B The effects of light, fertilization, plant density on Arbuscular mycorrhizal fungi in root of Compositae poisonous herb Ligularia virgaurea Huyuan Feng*, Guoxi Shi, Yongjun Liu, Shengjing Jiang, Lin Mao, Gang Cheng, Guozhen Du, Lizhe An School of Life Sciences, Lanzhou University, China

Arbuscular mycorrhizal (AM) fungi are ubiquitous in wide range of ecosystem, and form mutualism associations with roots of 80% plants. These mutualistic soil fungi received their carbohydrate from host plant in exchange for enhanced nutrient capture as well as improved host stress tolerance. Less evidences and theories about effects fertilization and light intensity on AM fungal richness and abundance are developed comparing to effects of these two factors on plant productivity and richness. In this study, we investigate that direct and indirect effects of light, fertilization, plant density on Arbuscular mycorrhizal fungi in root of Ligularia virgaurea. Both fertilization and light manipulation drive changes of soil properties and plant traits in opposite or same direction. Light manipulation had significant effects on intra- and extraradcial hyphae of AM fungi, and also fertilization affected only affected extraradical hyphae, but none of main effects of two factors(light and fertilization) affected spore density of AM fungi. A total of ten 10 morphological species of AM fungi were isolated from this study, including five species within the genus Glomus, one species from Entrophospora, and four species of Acaulospora. Both Acaulospora laconosa and Acaulospora gerdemelli were found (Frequency=100%) in all samples, but the latter is the most abundant AM fungal species (Relative abundance=21%) in all treatments. Fifty phlotypes of AM fungi were delimited by Mothur in all roots samples, based on 1571 positive clones. The results showed that fertilization reduced AM fungal abundance and richness by releasing resources and decreasing root:shoot, fertilization and shading accelerate jointly loss of AMF abundance and richness due to less biomass allocation to roots.

Acknowledgments: The research was supported by National Basic Research Program of China (2012CB026105), National Natural Foundation of China (40930533, 31170482), State Key Laboratory of Frozen Soil Engineering, Chinese Academy of Sciences (SKLFSE200901), and PhD Programs Foundation of the Ministry of Education of China (2010021111002, 20110211110021).

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106B Bacterial and Biochemical indicators of agricultural management for soil under no-till crop production Eva Figuerola1, Leandro Guerrero1, Alejandro Ferrari2, Silvina Rosa1, Leonardo Erijman1, Luis Wall*2 1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI- CONICET), Argentina, 2LBMIBS - DCyT - University of Quilmes, Argentina

The increasingly use of no-till agriculture, adopted in many areas of the world as an alternative to conventional farming, may contribute to reduce the erosion of soils and the increase in the soil carbon pool. However, the advantages of no-till agriculture are jeopardized when its use is linked to the expansion of crop monoculture. The aim of this study in a frame of a multidisciplinary project was to survey bacterial communities and Fatty Acids Methyl Ester profiles to find indicators of soil quality related to contrasting agriculture management in soils under no-till farming. Four sites in production agriculture, with different soil properties, situated across a west-east transect in the most productive region in the Argentinean pampas, were taken as the basis for replication. Working definitions of Good Agricultural Management and Poor Agricultural Management were adopted for two distinct scenarios in terms of crop rotation, fertilization, agrochemicals use and pest control. Non-cultivated soils nearby the agricultural sites were taken as additional control treatments. Tag-encoded pyrosequencing was used to deeply sample the rDNA from bacteria residing in soils corresponding to the three treatments at the four locations. Although bacterial communities as a whole appeared to be structured chiefly by a marked biogeographic provincialism, the distribution of a few taxa was shaped as well by environmental conditions related to agricultural management practices. A statistically supported approach was used to define candidates for management-indicator organisms, subsequently validated using quantitative PCR. Using a different approach, principal component analysis of Whole Cell Fatty Acids showed a clear separation of natural sites from the agricultural sites, and also a strong separation by geographical location of the natural sites. The Neutral Lipids Fatty Acids profile was the most revealing for differences in the structure of fatty acid composition concerning agricultural managements. The most relevant fatty acid groups for land use discrimination were mono- unsaturated, poly-unsaturated, straight chain and methylated ones. The neutral lipid fatty acid 20:0 show higher values in Good Managements than in Poor ones, while the fatty acid 18:1w7c was increased in Poor Managements compare to Good ones. Based on these results we propose two potential management indicators to discriminate between sustainable vs. non-sustainable agricultural practices in the Pampa region: 1) the ratio between the normalized abundance of a selected group of bacteria within the GP1 group of the phylum Acidobacteria and the genus Rubellimicrobium of the Alphaproteobacteria and 2) the ratio between the normalized abundance of 20:0 and 18:1w7c. Interestingly, there is consistency between both indices, in that fatty acid 18:1w7c is the most abundant membrane fatty acids in bacteria of the genus Rubellimicrobium, the potential marker for Poor Agricultural Managements.

107B Bacterial Mobilization of Sulfur and Phosphorous in Biochar Amended Soils Aaron Fox1, Ruth Cullen1, Witold Kwapinski2, Achim Schmalenberger*1 1University of Limerick, Life Sciences, Ireland, 2University of Limerick, Chemical and Environmental Sciences, Ireland

Biochar amended soils have demonstrated various plant growth promotion effects, increased water retention and a neutralized pH when applied to acidic soils. The char's impact, however, on macronutrient bio-availability and the microorganisms involved in their transformation remain largely elusive. This is particularly the case with sulfur (S) and phosphorous (P). Plants rely on microorganisms to mineralize organic and inorganically bonded S and P which the plant can then readily utilise. The aim of this study was to investigate S and P mobilizing bacteria in biochar amended soil to better understand nutrient mobilization capabilities in Irish soils with biochar.

Cultivable desulfonating and phosphorous mobilizing bacteria were quantified, isolated and characterised from the soil and rhizosphere associated with Maize (Zea mays) in biochar (Picea sitchensis wood, pyrolysed at 600°C) amended soil in a pot experiment and a field site in County Limerick in 2011. Quantification of aromatic sulfonate desulfurizing (desulfonating) bacteria was carried out in minimal media (MM) with toluenesulfonate (TS) as sole S source in a most probable number approach (MPN), while phosphorus mobilizing bacteria were quantified on minimal media 18

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August plates with tri-calcium phosphate as the sole source of P (TCP), detecting P solubilisation through clearance of particulate P. Subsequent isolation of pure cultures that grew better with TS than without as sole S source or formed a halo on the TCP plates were identified through 16S rRNA sequence analysis. Desulfonating bacteria were also tested on TCP plates and P mobilizing bacteria were tested on MM with TS for desulfonating activity. A PCR assay of the phnJ subunit of the carbon-phosphorous (C-P) lyase pathway was conducted on the bacterial isolates.

MPN analysis of desulfonating bacteria identified higher levels of desulfonating bacteria in biochar amended soils and rhizosphere soil of maize than in biochar free controls. 16S rRNA analysis revealed a high diversity of desulfonating bacterial isolates associated to Rhizobium, Bosea, Pseudomonas, Polaromonas and Acidovorax. While the latter two genera have been previously identified as desulfonating bacteria, no alpha and gamma Proteobacteria have been isolated from soils to date utilizing aromatic sulfonates as sole S source. The dominant P mobilizing bacterial isolate is associated to Rhanella aquatilis. A a selection of desulfonating strains were identified, capable of mobilising particulate P and a subset of the P mobilising isolates were capable of desulfonating TS in minimal media. Some of the S and/or P mobilising isolates also showed evidence of the capability to mobilise P from phosphonates, possessing a copy of the phnJ subunit of the C-P lyase pathway.

Additions of biochar have proven in several studies to be beneficial to plant growth but until today very little is known about the potential beneficial role of bacteria colonizing the chars. Here we report the presence of aromatic sulfonate desulfurizing alpha and gamma Proteobacteria in soils for the first time. Increased numbers of bacteria in biochar amended soils suggest that S and P mobilizing bacterial colonizers could be beneficial for plant growth through enhanced nutrient mobilization.

108B How does the disruption of Pleurozium and Sphagnum moss carpets effect the microbial communities of black spruce in boreal forested peatlands Mark Fox*, Yves Bergeron, Pascal Drouin University of Quebec, Canada

The forests floors throughout the boreal forests of North America and Eurasia are covered with a thick moss layer comprising mainly of Sphagnum spp. and Pleurozium schreberi). These mosses affect the soil temperature, moisture, nutrients availably and ecosystem properties like net primary productivity and decomposition. Mechanical site preparation (MPS) is a technique used after trees harvesting to improve the survival and growth of planted tree seedlings.The goal of this study was to examine the effects of MPS on the microbial populations in the soils around the planted trees. The study was performed in the boreal-forested peat lands of Northern Quebec. The moss carpet was disturbed using hand held gardening tools to a depth of about 25 cm. Only the organic layer was disrupted and the organic and mineral layers were not mixed. Both Sphagnum and Pleurozium carpet were disturbed in an attempt to find out if there was a difference in the soil microbes community between the two moss types. Bacterial community profiles were examined using BIOLOG Eco-plates and PCR-denaturing gradient gel electrophoresis was used to determine species diversity of both bacteria and fungi. BIOLOG Eco-plates patterns were significantly different between the disturbed pleurozium and disturbed sphagnum (53% similarity). Similar results were observed with the non-perturbed Sphagnum and Pleurozium soils (57% similarity). There was no significant difference between the disturbed and non-disturbed Pleurozium soils (98% similarity), while when the Sphagnum soils were perturbed a similarity shift to 65% was observed. Perturbation of Sphagnum had a greater effect on bacterial community than Pleurozium, possibly due to the fact that sphagnum decomposed more rapidly than Pleurozium after perturbation.DGGE analyses showed that there is significant difference in the diversity observed in the Pleurozium vs Sphagnum and not perturbed vs perturbed soils. High severity fires have been shown to promote the establishment of productive post disturbance stands black spruce forests of Northwestern Quebec while low intensity fires have been shown to produce less productive stands. It is believed that some MPS techniques are able to expose the mineral soil, suggesting that MSP could, to some extent, reproduce the effects of a high-severity fire to expose mineral soil. We have shown that site preparation does effect the microbial communities in these forests and could help promote the growth of black spruce stands.

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109B Microbial activity and plant community recovery from grazing may be mediated by nutrient availability in low Arctic meadows Gaia Francini*1, Minna-Maarit Kytöviita1, Sari Stark2, Minna Männistö3 1University of Jyväskylä, Finland, 2University of Lapland, Finland, 3METLA, Finland

Microbial communities and microbial activity is known to be affected by grazing; especially in the Arctic grazing by large ungulates is hypothesized to be the main factor shaping plant communities and affecting soil processes. Grazing in the tundra is supposed to induce a shift into the plant community from a moss dominated stage to a graminoid dominated one, improving soil nutrient cycling and favoring the fast bacterial-based energy channel over the slow fungal-based one. Still the experimental evidence shows very idiosyncratic responses, and results are context dependent. Furthermore, there is very little information on how the plant and soil communities respond to cessation of grazing and how the system changes afterwards.

In this study we address the question of how a long term relief from reindeer grazing affects soil microbial processes in two low Arctic meadows.

Our two study sites are located in the Kilpisjärvi region in Jehkas (69°05’N, 20°47 E) and Saana (69°03’ N, 20° 50’ E). Both sites are south exposed low Arctic meadows dominated by the grass Deschampsia flexuosa. A fence has excluded reindeer grazing activity in part of the meadows for 10 years. Plant biomass, plant community and soil cores were sampled both inside (no grazing for 10 years) and outside (grazed for the last centuries) the fenced areas. Soil microbial community structure was assessed using phospholipid fatty acid extraction. Soil microbial activity was assessed measuring acid-phosphatase, phenol oxidase, β-glucosidase, chitinase and protease activity. Soil parameters, + such as soil organic nitrogen, microbial nitrogen, NH4 , moisture, pH and organic matter were recorded as well.

Plant community structure was affected by site and exclosure; exclosure had a stronger effect in the nutrient rich site Jehkas. Fencing or site did not affect significantly plant biomass or Shannon’s diversity index. Organic matter content was lower inside the exclosure and in Jehkas compared to Saana. Only in Jehkas excluding grazers resulted in higher microbial nitrogen, organic nitrogen and acid-phosphatase, phenol oxidase and protease activities. In contrast, only in the Saana site, chitinase activity was positively and b-glucosidase negatively affected by exclosure. Microbial community structure was not affected by the site or by the exclosure.

Our results confirm that arctic microbial communities are resilient to environmental changes. Relief from grazing exerts a strong effect on microbial processes in terms of organic matter depletion and enzymatic activity. Grazing increases soil organic matter and reduces microbial activity probably due to mechanical compression of the soil, changes in root exudation and changes in litter quality as result of variations in vegetation composition. This effect is conditional to site fertility and within the time frame of the present study, only the nutrient rich site showed differences after cessation of grazing.

110B Treated waste water irrigation affects the soil microbiome seasonal dynamics Sammy Frenk*1, Yitzhak Hadar2, Dror Minz1 1Agricultural Research Organization - Volcani Center, Israel, 2The Hebrew University, Israel

Agricultural irrigation with treated wastewater (TWW) is a common and increasing practice in arid and semi-arid regions, and is used as an alternative to freshwater (FW). However, irrigation with TWW, which may contain dissolved organic matter, salts and microorganisms, may alter the total microbial community composition and function. The soil microbial community is responsible for providing various environmental services. Consequently, changes that could occur to the soil microbial community can affect soil functions and fertility. This work characterized the effects of irrigation of agricultural soils on community composition by the extraction and analysis of DNA and rRNA from these soils. The microbial composition was studied by next generation sequencing using tag-encoded FLX amplicon pyrosequencing (bTEFAP) approach of the PCR amplified 16S gene, 16S rRNA and amoA gene. Soil community composition data was compared to soil chemical and physical parameters and to the rate of four microbial activities previously described as related to soil fertility. The soil community composition was highly similar in different soil samples obtained at the end of winter (rain season) 20

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August regardless of the summer irrigation type. However, while community composition in FW irrigated soils slightly shifted during the summer irrigation season, the community in TWW irrigated soils shifted to a much greater degree, especially when active community was analyzed (based on cDNA obtained from 16S rRNA). The ammonia oxidizing bacterial community reacted less to seasonality but in a much greater degree to TWW irrigation. Further, few ammonia oxidizing bacteria operational taxonomic units (such as those related to Nitrosomonas nitrosa) were almost exclusively affiliated to soil irrigated with TWW. Despite the change in composition, the community structure was moderately affected, as diversity indices changes were insignificant. The bacterial composition of the active community was extremely different than that of the total community in all treatments. While the active community included mostly representatives from the Proteobacteria and Actinobacteria phyla, suggesting their importance in this environment, the total community was much more diverse. The results reported here are based on two long term experiments in two different soil types and climatic regions. We concluded that the microbial communities in Mediterranean and semi arid agricultural soil are highly influenced by seasonal irrigation and the use of TWW increases these changes. Finally, these results show the anthropogenic impact imposed to the microbial community in soil by TWW irrigation are sustainable in a multiannual time scale as the soil bacterial community returns to a similar composition every rainy season despite the irrigation type applied.

111B Soil bacterial community composition, activity and size disruption by metal oxide nanoparticles Sammy Frenk*1, Tal Ben-Moshe2, Ishai Dror2, Brian Berkowitz2, Dror Minz1 1Agricultural Research Organization - Volcani Center, Israel, 2Weizmann Institute of Science, Israel

Materials in the nanometer scale are reported to be found naturally in various environments, including volcanic dust, soil, fresh water and oceans. While these materials can be harmful to living creatures they are ubiquitous in the environment. On the other hand, manmade engineered nanoparticles (ENPs) are relatively new and their effects on the environment are currently unknown. Further, the increase in manufacturing of ENP-based products and their increased availability to the consumer market will eventually result in increased release of ENPs to the environment. These materials may find their way to the soil environment via wastewater, dumpsters and other anthropogenic sources. Because the soil bacterial community acts as one of the major service providers for humankind, for example in terms of agricultural production, water availability and organic matter mineralization, it is critical to study the effects of their exposure to ENPs. Metal oxide nanoparticles comprise one group of ENPs with many applications and characteristics, which could be potentially hazardous to the environment. In this work the soil bacterial community was evaluated for its susceptibility to two metal oxide ENPs, copper oxide (CuO) and magnetite (Fe3O4), both with size range . The well-being of the soil bacterial community was evaluated in terms of its activity, composition and size using both molecular methods and enzymatic assays. Two different soil types were evaluated, one sandy soil and one clay soil. In addition, the effect of these ENPs on assay accuracy was examined, as these materials may chemically alter the soil organic matter. Results indicate that CuO has a strong effect on bacterial community activity, composition and size in the sandy soil, it affects only the activity and composition in the clay soil. On the other hand, Fe3O4 affects the bacterial community activity and composition only in the sandy soil, with no visible effects on the community in clay soil. ENPs reduced bacterial community diversity, as measured by the richness and even distribution of operational taxonomic units (97% sequence similarity). The composition of the bacterial community was modified significantly, and specific groups of gram positive bacteria, in particularly Bacilli members, were affected by the ENPs.

To conclude, toxicity of ENPs to soil bacterial community was demonstrated for both types of metal oxides although it had a strong dependence on soil type; CuO was much more toxic and community in heavy soil was more resilient to both ENPs.

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112B Succession of nitrogen-cycling microbes in early volcanic environment on the island of Miyake (Japan) revealed by metagenomics Reiko Fujimura*1, Seok-Won Kim2, Yoshinori Sato3, Wataru Suda2, Tomoyasu Nishizawa1, Kenshiro Oshima2, Masahira Hattori2, Takashi Kamijo4, Hiroyuki Ohta1 1Ibaraki University, Japan, 2The University of Tokyo, Japan, 3Center for Conservation and Restoration Techniques National Research Institute for Cultural Properities, Japan, 4University of Tsukuba, Japan

New terrestrial substrates such as lava, tephra and volcanic ash, contain negligible amount of organic carbon and nitrogen. Our previous study revealed that chemolithoautotrophic iron-oxidizing diazotrophic bacteria, Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans were pioneer microbes in recent volcanic ash deposits (Fujimura et al., 2012). Here we investigated how the succession of nitrogen-cycling microorganisms occurs in early soil ecosystem with deposit age by metagenomic analysis. The study site (site OY) was established near the new crater formed by eruption of Mt. Oyama in 2000. Volcanic ash deposits taken in 2004, 2007 and 2010 (deposit age, 3.5- , 6.6- and 9.5-years old, respectively), were acidic (pH3.0-3.9) and contained trace amounts of total carbon and nitrogen (<0.1 to 0.3 g kg-1). DNAs were extracted from the deposits and pyrosequenced with 454 GS FLX Titaniumn system. After the filtration of redundant reads by 454 replicate filter, open reading frames (ORFs) were predicted using MetaGeneAnnotator. Predicted ORFs were annotated in KEGG (Kyoto Encyclopedia of Genes and Genomes) database to extract of nitrogen (N) -cycling protein coding sequences (CDSs), i.e., NifH, AmoA, NarG, NirS/K, NorB and NosZ. Further, the extracted CDSs were analyzed by BLASTP searches using our determined L. ferrooxidans C2-3 (Sato et al., 2009; Fujimura et al., 2012) genomic database and the NCBI non-redundant (nr) database for analysis of N-cycling microbial communities. We obtained 598,685, 253,003 and 1,213,490 of unique reads, and 96,999, 292,431 and 1,350,554 of predicted ORFs from the 3.5-, 6.6- and 9.5-years old deposits, respectively. The 29-31% of predicted ORFs were assigned to the known CDSs of KEGG database at a cutoff e-value of 10-5. The ratios of NifH and NarG in total predicted ORFs were higher than those of AmoA, NirS/K, NorB and NosZ in all the samples. The Acidithiobacillus and Leptospirillum sequences accounted for 42 and 41% of the total NifH, respectively, in the 3.5-years old deposit. By contrast, the sequences of heterotrophic bacteria such as Beijerinckia, Burkholderia, Herbaspirillum accounted for 41-46% in 6.6- and 9.5-years old deposits. The NarG sequence of Methylocella was the most abundant in 3.5-years old deposit (14%), but its percentage decreased in 6.6- (3%) and 9.5-years old deposits (1%). Instead, the NarG sequence of Thiomonas accounted for 30% and 22% in 6.6- and 9.5-years old deposits, respectively. In conclusion, our metagenomic analysis revealed the succession of N-cycling microbial communities in response to deposit age in the early soil ecosystem.

113B Diversity of Desulfonating Bacteria in the Mycorrhizosphere Jacinta Gahan*, Achim Schmalenberger University of Limerick, Life Sciences, Ireland

Sulfur (S) is an essential macronutrient which in soil is ~95 % organically bound. Two major forms of organo-S exist: sulfate esters and sulfonates. Many bacteria and fungi are capable of hydrolysing sulfate esters with sulfatases to release S. In contrast, a multi enzyme complex is necessary to utilize sulfonates, the dominant source of organo-S in soils. This desulfonation reaction is catalysed by specific bacteria. However, fungi may be important for the desulfonation process as well. Mycorrhizal hyphae provide an intimate link between the extended soil environment and the plant root. Indeed, for 80-90% of all land plants, growth is dependent on symbiotic mycorrhizal fungi and their bacterial colonizers. The aim of this study was to analyze both the magnitude and diversity of the bacterial soil community associated with mycorrhizal hyphae that are involved in sulfonate desulfurization.

Hyphae were picked from grass roots of an uncultivated grassland site using a light microscope at 100 times magnification. Bacteria were extracted from the hyphae and a bulk soil control using a series of sieves with decreasing pore size (710 µm, 500 µm, 212 µm and 64 µm) followed by a sucrose gradient centrifugation of the sievings from the 212 µm and 64 µm sieves. Quantification of cultivable heterotrophic bacteria based on Colony Forming Unit (CFU) count on R2A of the bacterial population on/in hyphae and bulk soil resulted in 106 CFU/g and 105 CFU/g, respectively. The morphologically distinct bacterial colonies were separated out into pure cultures on minimal media solidified with 22

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August agarose with toluenesulfonate as sole sulfur source (MM2TS). Bacteria that grew advantageously on MM2TS were transferred in triplicate into microtitre plates containing liquid MM2TS and a corresponding sulfur free minimal media. Optical density measurement at 590 nm identified 53 isolates with greater relative growth in MM2TS (P ≤ 0.05). 16S rRNA gene based Restriction Fragment Length Polymorphorism (RFLP) analysis was undertaken to ascertain the taxonomic diversity of isolates with significantly greater growth in MM2TS. RFLP identified 36 taxonomically distinct isolates, of which, 31 were cultured directly from hyphae and 5 from bulk soil. Cultivation independent community analysis based on Denaturing-Gradient-Gel Electrophoresis (DGGE) of DNA extracted directly from the 212 µm and 64 µm sievings of the hyphae and bulk soil indicated greater relative 16S rRNA gene diversity in the hyphal bacterial community. The hyphal associated bacterial community resulted in a DGGE band number of 29 and 21from the 212 µm and 64 µm sievings, respectively, while the bulk soil equivalent resulted in a DGGE band number of 14 and 4, respectively.

The results suggest that mycorrhizal hyphae are host to a comparatively large, taxonomically diverse community of bacteria partially involved in the mineralization and bio-availability of sulfonate-S in soil. These findings place an emphasis on mycorrhizal hyphae and bacterial associations that requires further investigation.

114B Pseudomonas putida constructs for buried landmine detection Shany Gefen-Treves*, Sharon Yagur-Kroll, Shimshon Belkin Hebrew University of Jerusalem, Israel

Over the last decade, there were almost 7000 landmine deaths and casualties per year worldwide. The global cost of landmine clearance and unexploded ordnance (UXO) using current technologies is estimated by the UN in the range of 30 billion dollars and hundreds of years of work. A microbial detection system may offer an elegant, highly sensitive and cost effective approach to address this challenge. We attempt to apply genetically engineered microbial biosensors, tailored to sense trace signatures of explosives and their degradation products (2,4,6-Trinitrotoluene [TNT] and 2,4- Dinitrotoluene [DNT]). Upon exposure, the presence of the target chemical induces a sensing element, namely a promoter region, fused to a reporting element which provides a dose-dependent measurable signal.

With the premise that soil bacteria have evolved to sense and exploit various compounds encountered in their microenvironment, we set to reveal novel sensing elements in this microbial habitat. Pseudomonas putida, a nutritional opportunistic organism and aromatic compounds-degrading soil bacterium, represents a potentially promising platform to address this challenge. We constructed a mini-transposon based library, comprising mutants bearing promoter-less reporter bioluminescence genes (Vibrio harveyi, luxAB) at random insertions across the P. putida (KT2442) genome. A scan of the library for responses to 2,4-DNT yielded several potentially useful constructs. The characterization of these candidates is described, as well as the first optimization steps of their potential for rapid detection of 2,4-DNT vapors.

115B Eukariotic metatranscriptomes from two contrasted alpine meadows Roberto Geremia*, Tarfa Mustafa, Lucie Zinger, Philippe Choler, Eric Coissac, Jean-Marc Bonneville Université Grenoble 1, CNRS, France

In temperate-latitude alpine tundra, plant communities are distributed along a strong snow cover gradient. At the extremes of this gradient, early snow melt meadows harbor slow growing, stress- adapted plant species; while late snow melt meadows contain fast-growing, exploitative plants. Bacterial and fungal communities from these two meadows also strongly differ during plant growing season in terms of both community composition and phylogenetic structure, suggesting a strong influence of abiotic conditions and plant cover. How these biotic and abiotic contrasts affect the metabolic capacity and diversity of microbial communities remains however largely unknown. To gain insight on this point, we studied the metatranscriptomes of soil eukaryotic communities in these two alpine meadows.

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The cDNA from each soil was obtained from PolyA+ RNA, amplified by PCR and sequenced using massif parallel pyrosequencing. Around 80 000 reads per soil were obtained, rendering each ~40000 putative coding reads of, ~ 400 nt in length after read trimming and rRNA reads removal.

By using a combination of SwissProt, Blast2GO and MG-RAST annotation pipelines, we succeeded in identifying 38% of the reads as proteins with known function. Between 600 and 800 enzymatic functions were listed for each soil, and Fungi were found as the most abundant group, followed by plants and animals.

We further observed that both ribosomal proteins and ubiquitin ligases were underrepresented in early snow melt soils, suggesting a decrease in protein turnover. Interestingly, glucose utilization also to diverged between the two habitats. In the early snowmelt soil, eight functions associated with glycolysis and Kreb’s cycle were underrepresented, while three functions associated with polysaccharide metabolism are overrepresented. These results suggest that early snowmelt site eukaryotic communities hydrolyses chiefly cellulose and converts part of the resulting glucose towards stress-related molecules, and display therefore a conservative strategy. In contrast, late snowmelt soil eukaryotic communities rather display a resource exploitative feature, by producing glucose from starch hydrolysis and using it through glycolysis and Krebs cycle.

These observations at the mRNA expression level therefore suggest that soil micro-eukaryotes display similar traits that the aboveground plants.

116B Comparison of bacterial numbers and carbon usage patterns in tree root zones between copper mine tailing sites and control sites in the keweenaw peninsula of michigan Timothy Gsell* Governors State University, USA

The Keweenaw region of the Upper Peninsula of Michigan is known for copper mining over the past two centuries. Although there are no active mining operations today, the remnants of past mining practices remain. Large amounts of mine tailings and stamp sands sit in piles all along the ridgelines that run the entire length of the Keweenaw Peninsula. The Estivant pines sanctuary, near Copper Harbor MI, exists among these abandoned mine sites as an island of old growth Eastern White Pines and other species of trees. Root zones from these trees are stable environments rich in nutrients and supporting higher microbial numbers and diversity than areas without root infiltration. Microorganisms in this study were compared in mine site tree root zones from both Cliff and Delaware mines and compared to that of the control counterparts in the Estivant Pines Preserve. It was hypothesized that residual mine practice contamination from arsenic and copper that reach 600 ppb, may drastically effect the numbers and diversity of root zone bacteria which may in term effect the health of the tree populations and ecosystem. This may also be due to lack of other important resources, especially carbon, which appear to be limited in the tailing piles. A polyphasic approach was employed to compare microbiological results from these soil samples. The analyses performed were aerobic heterotrophic plate counts, direct microscopic enumeration, colony morphology-based diversity indices and BiOLOG physiological fingerprinting. BiOLOG analysis included both the use of GN and GP identification micro-plates and the three panel Eco-plates used to describe the bacterial community carbon usage at the various tree root zone sample sites. BiOLOG GN/GP plate analysis indicated Actinomycete genus types in nearly all samples. Various species of Rhizobium and Bacillus genus were also found in sites in several mine and control samples. Bacterial total direct counts were at or above 1 x 10^8 cells/g sediment except for several root zones in both mines, where cell numbers peaked at low 1 X 10^7 levels and were as low as high 1 X 10^5. Culture based counts were often two to three orders of magnitude lower than their respective total direct counts, although patterns between root zones were similar. Trends show microbial numbers dropped consistently in mine site soil with decreasing % culturablity with nearly all tree species. This suggests a larger proportion of microbes in the mine site samples potentially consist of distinct bacterial types compared to near surface sites from control trees. A principal components analysis of the 31 potential carbon sources found in the BiOLOG eco-plates show the microbes associated with mines group together, irrespective of the mine root zones, except for the Paper Birch samples. This was the only tree type found directly on the tailing piles and stamp sands on Lake Superior's shore. It was thought that this may be the result unique 24

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August fungi, or the redox of metals and chemolithoautotrophic activity providing carbon sources not available, or at different ratios, than in the Estivant site samples.

117B Is alginate the only "super-star" among the Pseudomonas putida exopolysaccharides under water stress? Gamze Gulez*1, Arnaud Dechesne1, Tim Tolker-Nielsen2, Barth F. Smets1 1Technical University of Denmark, Denmark, 2Copenhagen University, Denmark

Water availability to bacteria living in soil changes frequently due climatic events or agricultural activities, thus soil bacteria often face water stress. They, however, are very well equipped with diverse tolerance mechanisms to cope with such stress. One of the major tolerance mechanisms is the production of exopolysaccharides. Pseudomonas putida KT2440 can produce a diverse set of polysaccharides (alginate, cellulose, putida polysaccharides a and b). Alginate is considered the main one. Alginate is suggested to play a role in maintaining hydrated microenvironment upon drying so that bacteria can survive and gain time to adapt the changing environment. Our previous whole genome microarray study confirmed significant induction of synthesis genes for alginate, but not for other polysaccharide; strengthening the “super-star” role of alginate under water stress. But why are the other polysaccharides in the “cast” if they are not to be the “super-star”? Are there “hidden talents” of them waiting their turn to shine?

To answer these questions, we compared colonies of KT2440 wild type, alginate deficient mutant and a third mutant deficient of all known polysaccharides under water stress and non-stressed conditions. We grew each strain on the Pressurized Porous Surface Model which can control water availability as observed in soil pores. The colony images captured by epifluorescence stereo microscopy were analyzed for statistical variation between morphological parameters (fractal dimension, roundness, perimeter, aspect ratio, area). RNA from replicates of colonies was extracted to determine whole genome expression profile via microarray analysis. Colonies from the replicate setups were also sampled for EPS composition and quantification analysis.

Principal component analysis (PCA) indicated that all morphological parameters contributed almost equally to the variation observed among samples. Both PC1 (47.5 % variation) and PC2 (28% variation) grouped alginate and wild type colonies opposite to the colonies of the third mutant deficient in all known polysaccharides. This indicates that absence of any known polysaccharides may affect the colony morphology regardless of there is water stress or not. Moreover, according to PCA there was no separation between the wild type and alginate mutant colonies; both strains grouped together for each condition (that is water stress and no-stress).

Our preliminary results suggest that polysaccharides may also have functions under non-stressed conditions as overall polysaccharide mutant had similar morphologies both under water stress and non-stressed conditions which was distinct from the wildtype. More importantly, our results suggest that under water stress, absence of alginate could be compensated by other polysaccharides as alginate mutant showed similar morphology to wild type colonies. Once the microarray and EPS composition data were revealed, we would be able to say more and see if other polysaccharides are definitely playing a role under water stress.

118B The role of sugarcane managements on the structuring of fungal communities in soils Thiago Gumiere*1, Ademir Durrer Bigaton2, Mayra Camargo Andrade Costa3, Fernando Dini Andreote3 1ESALQ/USP, Brazil, 2“Luiz de Queiroz” College of Agriculture/University of São Paulo, Brazil, 3Queiroz ”College of Agriculture/University of São Paulo, Brazil

The cultivation of sugarcane has been showing undeniable importance in the agriculture of the São Paulo State (Brazil), where the agricultural zoning describes the existence of regions with different patterns of environmental conditions suitable for this crop. In this environment, high heterogeneity of cultivation processes and wide differences of soils result in the presence of a highly diverse microbial communities, which live and work together to maintain the functionality of the soil, thus interfering in the development of plants. Thus, this study aims to give a first glance on the roles of sugarcane managements on the residing diversity of fungi in soils cultivated with this crop. Within a production 25

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August unit, eighteen cultivation fields with distinct managements were selected, and three samples were collected in each of them, encompassing the soil layer 0 to 15cm. Soil cores were subjected to chemical and physical characterization, and also used for fungal fingerprinting by fungal ITS-based PCR-DGGE. The non-metric multidimensional scaling (nMDS) based on PCR-DGGE patterns revealed the presence of five clusters encompassing the eighteen areas. The combination of fungal fingerprinting with physical, chemical and management data, made by principal components analysis (PCA), does indicate as the majors factors involving in the formation of clusters, like as the pH, the contents of organic matter, the available phosphorus, and the number of the harvests performed in the area, explaining 11, 6, 9 and 4% of variance, respectively. More specifically, assessing each factor separately, it was observed also a negative effect on the fungal richness attributed to the vinasse addition in soils, while the expected variation promoted by soil granulometry was absent (clay soil or sandy soil shared same richness numbers). The role of vinasse might be linked with a selection event, promoted by the high input of specific nutrients in soils, as for example, potassium; while physical characteristics of soils does not alter the total number of fungal species, but may be shifting the occurrence of specific groups, what is still to be explored. In summary, we provide here a first stone on how distinct managements in sugarcane fields can change the composition of microbial communities residing in such soils, what can be further explored in order to support the development of a more sustainable production system for this crop.

119B Seeping volcanic CO2 and its effects on freshwater environments Simone Gwosdz*1, Ingo Möller1, Hans Richnow2, Martin Krueger1 1Federal Institute for Geosciences and Natural Resources, Germany, 2Helmholtz Centre for Environmental Research, Germany

The Laacher See volcanic centre, located in the middle of the East Eifel volcanic field, west of the river Rhine, discharges about 5000 t CO2 per year. The CO2 is released from multiple gas vents, at the bottom of the lake. Natural CO2 sources like Laacher See, allow the determination of CO2 induced biological and geochemical alterations of surface ecosystems. Therefore, the Laacher See is a suitable natural analogue for possible leakages from potential carbon dioxide storage sites (CCS). CO2 capture and subsequent storage in deep geological formations is one option currently investigated to reduce the crease of atmospheric greenhouse gas concentration caused by fossil energy consumption.

To assess effects of elevated CO2 concentrations on freshwater sediment ecosystems, we studied microbial activities, abundance and diversity together with related biogeochemical parameters. CO2 seeps at the lake bottom and potential control areas were located using several hydroacoustic measurements. The flux rates and composition of rising CO2 were verified with divers and a small remotely operated vehicle (ROV).

Dissolved CO2 in bottom water as well as sediment porewater samples had a carbon isotopic signature close to that in the gas bubbles, confirming a magmatic origin of the gas. Analysis of water samples collected close to intensive CO2 seeps showed low pH and an increase of dissolved CO2,especially at the end of the summer stratification period. Furthermore, geochemical and microbiological analyses of up to 2m long sediment cores from CO2-affected and control sites showed alterations in pH, microbial activity and populations.

Our results illustrate a CO2 induced shift in microbial activity and community composition caused by the increasingly anaerobic and acidic environmental conditions.

120B Impact of simulated climate change scenarios on extracellular enzyme activity of soil microbes Verena Hammerl*1, Kerstin Grant2, Jürgen Kreyling2, Anke Jentsch2, Karin Pritsch3, Michael Schloter3 1TU München, Germany, 2University of Bayreuth, Germany, 3Helmholtz Zentrum München, Germany

Prolonged climate change like extreme drought events during vegetation periods or winter warming as predicted in future climate scenarios will affect ecosystem functions in multiple ways. Increased 26

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August frequencies of soil freeze-thaw cycles resulting from winter warming lead to an increase in nutrient release due to physical disruption of soil aggregates and humic materials, and consequently, also affects soil microorganisms. Further on, long lasting drought events affect not only plants but again also soil microbes. Important soil functions, like nutrient turnover processes will be changed by both climate change scenarios. These factors are addressed in the EVENT-Experiment established at the Botanical Garden of the University of Bayreuth and focuses on natural grassland communities.

In this project, we hypothesise that under drought conditions and freeze-thaw cycles hydrolytic enzyme activities will be reduced. Therefore, biochemical parameters such as soil potential extracellular enzymatic activities (PEEA) of MUF-labled hydrolytic enzymes (e.g. phosphatase, chitinase, cellulases), changes in the carbon content and microbial biomass were measured.

In three consecutive sampling years seasonal differences in potential extracellular enzyme activities between spring drought and summer drought treated soils were determined. Whereas after both treatments significantly decreased PEEA values were measured between drought treated and control soils, PEEA increased notably within two weeks after rewetting of summer drought treated soils due to a higher amount of available biomass. PEEA values of spring drought treated soils recovered only to the level of the control plots. Our first results in winter warming showed no consistent treatment effects.

121B Soil fungal and bacterial communities respond differently to long-term climate change and their responses are consistent despite seasonal variations in temperature and water availability Merian Haugwitz*, Lasse Bergmark, Anders Michelsen, Anders Priemé University of Copenhagen, Denmark

Soil microbial communities are important determinants of ecosystem processes. It is therefore essential to understand the impact of climate change on these communities for predicting how ecosystems will respond to future environmental conditions. In a long-term climate change experiment the effects of increased temperature (on average 0.3 °C at 5 cm soil depth), elevated CO2 (510 ppm) and experimental pre-summer drought periods were investigated both solely and in combinations at a Danish temperate heathland dominated by heather (Calluna vulgaris) and wavy hair-grass (Deschampsia flexuosa). We combined high-throughput pyrosequencing and qPCR of 16S DNA and ITS fragments with biomass measures to characterise the climate change effects on soil bacterial and fungal communities during the fifth treatment year, and related this to seasonal fluctuations in soil temperature and soil water availability.

Overall, the abundance of fungi and bacteria were higher during spring and early summer (March and June) compared to autumn (November). Fungi quantitatively dominated the soil throughout the year and especially during spring the fungal to bacterial ratio peaked, a shift that was even further increased by the experimental pre-summer drought. Despite the annual fluctuations in the microbial communities, the slight soil temperature increase of 0.3 °C resulted in significantly higher abundance of fungi and bacteria, with fungi responding most consistent and strongly across seasons. Furthermore, the fungal community structure was affected, with increased temperature and drought resulting in higher abundance of saprotrophic fungi, and elevated CO2 in more symbiotic fungi. In contrast, the bacterial community structure remained relatively stable in the different climate change manipulations despite large seasonal changes in abundance.

Unexpectedly, there were no interactive effects of increased soil temperature, drought and elevated CO2 on fungal and bacterial abundance or community structure. Our results rather suggest that in the long-term, soil fungal and bacterial communities are most strongly controlled by temperature and drought as single factors, with temperature potentially having the highest impact on the soil ecosystem for example through changes in the relative abundance of functional groups of soil fungi.

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122B Effects of free-air CO2 enrichment (FACE) and soil warming on ammonia-oxidizing bacteria and archaea in flooded paddy soil Masahito Hayatsu*, Kanako Tago, Takeshi Tokida, Hirofumi Nakamura, Hidemitsu Sakai, Yasuhiro Usui, Kentaro Hayashi, Toshihiro Hasegawa National Institute for Agro-Environmental Sciences, Japan

The ever-increasing concentration of atmospheric CO2 is one of the major contributing factors associated with climate change and global warming. These impacts on soil microbial communities and activities are poorly understood and controversial. Rice fields are considered to be the most important agricultural ecosystems in Asia. Therefore, the free-air carbon dioxide enrichment (FACE) system of rice field was constructed to explore the response of soil microorganisms to climate change by artificially elevating atmospheric CO2 concentration. In flooded paddy field nitrification is a rate limiting step of nitrogen cycle. This study reports the effects of elevated atmospheric CO2 and soil warming on diversity and abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in paddy field.

The present study was conducted in 2011 as part of ongoing research of rice-FACE established in 2010 at Tsukubamirai, Japan. Four rice paddy fields were used as replicates, each with two areas 2 (ambient levels of CO2 (Amb) and enriched CO2 (FACE)). Each treatment area was a 240-m octagon -1 ring, and the CO2 level within FACE ring was set to 200 μmol mol above the ambient concentration by a pure CO2 injection system. Each ring included surface water and soil temperature treatments in a split-plot design. Temperatures were normal or elevated, with the latter targeted at 2°C above normal temperature using heating wires placed on the soil surface. The soil samples were taken from the Amb and FACE fields at depths of 0-1 cm (upper layer) and 1-10 cm (lower layer) at July in 2011. Nitrification potential was measured by the shaken soil slurry method. DNA samples were prepared by Fast DNA spin kit for soil. Community structure of AOA and AOB was analyzed by PCR DGGE of amoA amplicon. Abundance of AOA and AOB was determined by quantitative PCR targeting amoA.

The nitrification potential of upper layer soil was similar to that of lower layer soil. Elevated temperature and enriched CO2 did not affect the nitrification potential. DGGE proiles showed that the community structure of AOB appeared to be slightly influenced by enriched CO2. The community structure of AOA was not changed by enriched CO2. Elevated temperature did not affect the AOA and AOB community structure. The abundance of AOB of upper layer soil was significantly higher than that of lower layer soil in FACE and Amb. The abundance of AOA and AOB of lower layer soil was significantly lower in the elevated temperature plots than in the normal temperature plots. No statistically significant difference was observed in the abundance of AOA and AOB of upper layer soil in both temperature treated plots in FACE and Amb. The enriched CO2 did not have impact on the abundance of AOA and AOB. The results of the present study indicated that elevated temperature and enriched CO2 seemed to slightly influence AOA and AOB ecology. Our investigation was carried out only in 2011. Further continuous investigation is needed to determine the effects of elevated temperature and CO2 enrichment on AOA and AOB.

123B Sub-arctic alpine heath tundra has a more open and bacterial-dominated N cycle than adjacent mountain birch forest Maria Hellman*1, Karina Clemmensen1, Anders Michelsen2, Sara Hallin1 1Swedish University of Agricultural Sciences, Sweden, 2University of Copenhagen, Denmark

In sub-arctic Scandinavia, mountain birch forest is expected to expand into alpine heath tundra with climate warming. Our hypothesis is that this will result in a more closed nitrogen (N) cycle because of a more efficient nutrient-recycling from organic matter in ecosystems dominated by ectomycorrhizal trees than in heath tundra. As an indication of inorganic N-cycling, the abundances of the ammonia oxidizing and denitrifying microbial communities were analyzed along a heath-to-forest ecotone near Abisko, northern Sweden, using quantitative PCR of key enzyme coding genes specific for the functional groups. In addition, the bacterial to fungal ratio was estimated by qPCR of the 16S and ITS regions of the rRNA genes and soil pH and total and extractable carbon (C) and N pools were quantified. The humus layer was sampled at three depths in 24 plots representing four habitats (heath, shrub heath, forest line and forest) along the gradient. To assess if sampling plots within the defined habitats shared environmental features and if habitats differed from each other, samples were compared based on soil parameters using non-metric multidimensional scaling and multi-response 28

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August permutation procedures. All habitats were significantly (p<0.05) different from each other, except for shrub heath and forest line, and irrespective of habitat, the deepest layer was always significantly different from the upper layers. The variables contributing most to the separation of samples according to habitat were dissolved organic C (DOC), DON, C:N, all high in the forest and low in the heath, and + pH and NH4 , which were high in the heath and low in the forest. Interestingly, archaeal ammonia oxidizers could not be detected in any of the habitats, suggesting that ammonia oxidation was driven solely by the bacterial counterpart. Abundances of genes reflecting the size of the denitrifier and bacterial ammonia oxidizing communities, and of total bacterial and fungal communities, expressed either per ng extracted DNA, per g soil, or per surface area, were significantly higher in the heath tundra than in the forested habitats. Nevertheless, the bacterial:fungal ratio decreased along the gradient, suggesting that bacterial communities were more important for nutrient cycling towards the heath tundra and fungi were more important in the forest. The ammonia oxidizing fraction of the total bacterial community did not display any specific trend along the gradient, but the denitrifying fraction was significantly larger in the heath tundra indicating that these habitats supported an increased - growth of denitrifiers. This coincided with decreasing DOC:NO3 ratios, which in theory would favor - denitrification over other NO3 reducing processes. Overall our results show that the soil N cycle is more open in the heath and support bacterial driven nutrient cycling with inorganic N transformations potentially leading to gaseous N losses.

124B Microbial communities associated with root-knot nematodes in soil Holger Heuer*, Mohamed Adam, Johannes Hallmann Julius Kühn-Institute, Germany

Root-knot nematodes (Meloidogyne spp.) are one of the most damaging crop pathogens in the world. The newly-hatched juveniles of this obligate parasite have a short free-living stage in the rhizosphere soil of the host plant where a high diversity of microbial species is present. Specific attachment of microbes to the cuticle of juveniles may play an ecological role, e. g. in transport of microbes through soil, microbial endophytic colonization or co-infection of roots, induction of plant defence systems, or microbial infection of the nematode. A better insight into nematode-microbe interactions might result in the discovery or improved application of biocontrol agents. The aim of this study was to assess by DNA-based techniques the species of bacteria and fungi that specifically attach to Meloidogyne incognita juveniles in soil. Bacterial and fungal communities of arable soil and that of juveniles extracted from this soil were compared by PCR-DGGE fingerprints of 16S rRNA gene or ITS fragments, respectively. Numerous nematode-specific microbial ribotypes were identified which were abundant on the nematode surface but not in the surrounding soil. Some bands from Fungi, Bacillus, Alpha- and Betaproteobacteria were consistently enriched on most replicate samples of juveniles. This was observed for four different races of M. incognita. In contrast, Pseudomonas spp., Actinobacteria, and Enterobacteria showed high variability among replicate juveniles and races, suggesting that species of these taxa were not specifically attaching to the nematode cuticle. Adverse effects of cultured soil bacteria on motility, survival, hatching, development, and reproduction of M. incognita could be shown in vitro (by testing the effect of bacterial culture supernatants on juveniles), and in pot experiments with tomato plants (by testing the reduction of root invasion through bacterial soil or seed inoculation). In conclusion, specific bacteria and fungi attached to the cuticle of M. incognita juveniles in soil and putatively affected the nematodes. Currently, we investigate whether similar species attach to the cuticle in three different arable soils, and how colonization of the cuticle relates to suppressiveness of the soils against root-knot nematodes.

125B Effect of three fungicides on degradation of diuron in tea field soil and impacts on soil Yu-hua Hsiao*, Zhi-Zhan Li, Jui-Hung Yen Department of Agricultural Chemistry, National Taiwan University, Taiwan

In this study, the effect of mix application of three fungicides, benomyl, tebuconazole and triadimefon, with herbicide, diuron, on persistent of these pesticides on soil was investigated. Meanwhile, the impact of mixed application on soil bacterial communities was also monitored. From the result, diuron degraded more slowly in sterilized soil than in unsterilized soil, it means that diuron degraded by biodegradation in tea field soil. In 10-fold field rate of herbicide and fungicides application treatments, diuron DT50 are 125 (Diuron only, D), 141 (Benomyl+Diuron, B+D), 127 (Tebuconazole+Diuron, Te+D) and 122 day (Triadimefon+Diuron, Tr+D). The B+D treatment decreased diuron degradation rate 29

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August

slightly. In 50-fold field rate treatments, diuron DT50 are 167 (D), 231 (B+D), 189 (Te+D) and 238 day (Tr+D). B+D and Tr+D treatments reduced diuron degradation rate significantly. The diuron metabolite N'-[3,4-dichlorophenyl]-N,N- methylurea (DCPMU) was found in 10-fold field rate treatments, DCPMU and N'-3,4-dichloro-phenylurea (DCPU) were found in 50-fold field rate in experimental period. Benomyl may inhibit the degradation of diuron, therefor the amount of DCPMU product in B+D treatment is less than other treatment in 50-fold field rate. D, Te+D and Tr+D applied in tea field may increase the bacterial community richness, and B+D impacted on the bacterial community by detected the amount of band on DGGE fingerprint and cluster analysis in 10-fold field rate. Tr+D and B+D impacted on the bacterial comunity in 50-fold field rate. The impact on bacterial community may inhibit the degradation of diuron and therefore increase the persistence of diuron in environment.

126B Diversity of acidophilic actinobacteria in red soils, and genomic characterization of a novel Streptomyces lineage Ying Huang1, Xiaoxuan Guo*1, Xiaoying Rong1, Xiaomin Li2, Yongsheng Gao2, Jisheng Ruan1 1Institute of Microbiology, Chinese Academy of Sciences, China, 2Jiangxi Agricultural University, China

Red soils are one of the typical agricultural soils in southern China. These soils have been subjected to intense weathering, and mineral nutrients (including Ca, Mg, and K) in the soil are mostly eluted, forming the major soil constraints of low organic carbon and alkali metal levels, high iron and aluminum oxides, and acidity. As such, the red soil seems to be a potential habitat of unique and diverse microorganisms, especially acidophilic actinobacteria, which are usually chemoorganotrophs, degrading complex polymeric substrates during their initial development and consuming stored carbon during sporulation. To investigate the acidophilic actinobacteria diversity and ecological characteristics in red soils, soil samples were collected from the Red Soil Ecology Experimental Station of Chinese Academy of Sciences at Liujiazhan (28°12′N, 116°55′E, pH 4.8), Jiangxi Province of China. Culture- independent method base on construction of 16S rRNA gene clone libraries with two Actinobacteria- specific PCR primer sets as well as conventional culture methods were employed. A total of 342 16S rRNA gene clones were sequenced, and 65.5% sequences fell into new taxa using a sequence identity value of 98%. The prediction of actual diversity using Mothur revealed 138 OTUs belonging to 8 suborders, 17 families and 39 genera within the class Actinobacteria. The majority of clone sequences were affiliated with families Geodermatophilaceae, Pseudonocardiaceae and Thermomonosporaceae. On the basis of the cultivation approach, 180 isolates were purified, which could be assigned to 11 families and 22 genera. Among the strains isolated, at least 20 new phylotypes were obtained, which appeared to represent 13 new taxa. These results show the existence of diverse populations of phylogenetically distinct actinobacteria in the red soils. Most isolates belong to the families Streptomycetaceae (60%) and Streptosporangiaceae (10%), of which streptomycetes composed the majority, and a cluster of novel streptomycetes with outstanding antifungal activities was discovered. The 16 representative strains of this cluster displayed diversity by Box-PCR fingerprints and physiological traits, but multi-locus sequence analysis supported the delineation of these strains as a new species. Genome analysis of Streptomyces sp. FXJ1.532, a representative of this novel lineage, revealed the presence of a large number of genes associated with inorganic ion transport (182/6,716) and metal/drug resistance (44/6,716), which might contribute to the organism's tolerance to various toxic compounds, implying the distinct physiological characteristics evolved via adaptation to the red soil habitat. The existence of several degradation enzymes, especially the identification of chitinase, agreed with the experimental evidence implying that this lineage may play significant roles in nitrogen and carbon cycling in the red soil. Furthermore, the presence of over 22% (1,498/6,716) hypothetical proteins with no match to any known proteins might reflect a high degree of specificity of this novel lineage, which worth continued exploration.

127B Metagenomic analysis uncovered novel structure of microbial communities involved in denitrification in paddy soil Hideomi Itoh*1, Satoshi Ishii1, Yutaka Shiratori2, Kenshiro Oshima1, Shigeto Otsuka1, Masahira Hattori1, Keishi Senoo1 1The University of Tokyo, Japan, 2Niigata Agricultural Research Institute, Japan

Denitrification is one of the important processes involved in biological nitrogen transformation in paddy soil. Dissimilatory nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide (N2O) 30

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August reductase (Nos) are key enzymes in denitrification. Diversity of genes encoding these enzymes (nir, nor, nosZ) in paddy soils have been investigated as markers of denitrifying microbial community structure through PCR-based analyses. However, PCR-based analysis is not enough to reveal whole diversity of these genes because of the limitation of PCR primer sequence. To overcome this problem, we obtained metagenomic sequence of rice paddy soil without any prior PCR amplification. In this study, diversity of nir, nor, and nosZ sequences in the soil metagenome was investigated. In addition, we isolated and characterized Anaeromyxobacter sp. bacteria frequently detected in the soil metagenome. Soil samples were collected from a paddy field in Japan before waterlogging and during waterlogged periods in rice growing season in 2009. Metagenomic sequencing of the each soil DNA was performed using GS-FLX titanium. All of the obtained unique sequences were compared against the each functional gene database containing reference sequences of nir, nor, and nosZ obtained from Functional Gene Pipeline/Repository. Hit sequences were compared against NCBI non-redundant protein database and phylogenetically analyzed based on the lowest common ancestor algorithms with MEGAN software. Additional metagenomic data of the Minnesota farm soil and the Puerto Rico forest soil were obtained from MG-RAST server and analysed in the same way as described above. Among a total of 1725K sequences in paddy soil metagenomic data, 75, 187 and 114 sequences were assigned to nir, nor, and nosZ, respectively. Large majority of nir fragments were related to nir of members of the phylum Alpha-, Beta-, and Gammaproteobacteria, and Verrucomicrobia. Surprisingly, we detected nor and nosZ fragments related to not only denitrifiers belonging to Alpha-, Beta-, and Gammaproteobacteria but also non-denitrifiers belonging to Acidobacteria and Deltaproteobacteria carrying no nir gene on their genome. Furthermore, more than 30% of nosZ fragments in the paddy soil metagenome were related to nosZ of Anaeromyxobacter bacteria belonging to the phylum Deltaproteobacteria (identity, 55-92%), which have rarely been detected by previous PCR-based analyses. The nosZ sequences related to that of Anaeromyxobacter were also found in the metagenomic data of the farm soil and forest soil. Alignment analysis against sequences of universal primers for the PCR amplification of nosZ indicated that previous PCR-based analysis could not detect these nosZ sequences related to Anaeromyxobacter because of primer mismatches. To isolate the Anaeromyxobacter bacteria from the paddy soil, a dilution of submerged paddy soil was spread on R2A agar containing 5 mM fumarate and anaerobically incubated. As a result, two strains of Anaeromyxobacter sp. were obtained. Both of them showed potential ability to reduce exogenous N2O to N2 under anaerobic condition. In conclusion, our metagenomic analysis revealed novel structure of microbial communities involved in denitrification which could not be detected by the previous PCR-based analysis. Anaeromyxobacter bacteria, non-denitrifying N2O reducer, might be an important player of N2O reduction in paddy soil.

128B Shifts in bacterial community structures along xenobiotic contamination gradients in soils of cold and temperate climates Irena Ivshina1, Victor Bezel2, Tatyana Zhuykova3, Valentina Zhuykova3, Ludmila Kostina4, Tatyana Kamenskikh4, Anastasiya Krivoruchko*1, Maria Kuyukina1, Lyubov Ogurtsova5, Andrey Soromotin5, Tatyana Moiseenko6 1Institute of Ecology and Genetics of Microorganisms / Perm State National Research University, Russia, 2Institute of Ecology of Plants and Animals, Russia, 3Nighny Tagil State Social-Pegagogical Academy, Russia, 4Institute of Ecology and Genetics of Microorganisms, Russia, 5Research Institute of Ecology and Natural Resources Managment of Tyumen State University, Russia, 6Vernadsky Institute of Geochemistry and Analytical Chemistry, Russia

Chemical contamination of the environment is dramatically increased affecting fragile sensitive soil ecosystems of cold and temperate climates. Bacteria play a key role in adaptation and self-attenuation of soil biotopes from xenobiotics. At that, responses of soil bacterial communities to xenobiotics at low temperatures are poorly investigated. The present work is aimed at territory-oriented studies on bacterial community structures along xenobiotic contamination gradients in soils of cold and temperate climates. Soil samples (168) were collected within 2009-2011 from rizosphere and 0-20 cm deep of soil profile near the oil production and metallurgical works during scaled expeditionary investigations in the northern and middle (N 55-78°, E 51-78°) areas of Perm, Sverdlovsk and Tyumen regions, Russia. Concentrations of hydrocarbons (alkanes, benzene analogues, polyaromatics) and heavy metals (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn) were determined by GC-MS and AAS. The total toxic load (Si) was calculated as ratio of toxicants present in soil and those in the background level. Rates of dead plant destruction by soil bacteria were measured in field experiments with prepared dried debris of legumes, 31

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August cereals or herbs introduced in soil in 5x10 cm perforated capronic bags. Functional diversity and total numbers of soil bacteria including heterotrophs, nitrogen fixers, ammonifiers, nitrifiers, denitrifiers, iron oxidizers, sulphate reducers, cellulose fermenting bacteria, and hydrocarbon degraders were detremined by growth on selective media, respirometry, light microscopy and species-specific PCR detection in situ. Isolates were identified by polyphasic taxonomic approach by phenotypic, biochemical, chemotaxonomic, immunochemical, and 16S rRNA gene sequencing methods Resistance of isolates to pollutants was determined by staining with iodonitrotetrazolium chloride. The significant changes in soil bacterial community structures after long-term xenobiotic contamination were shown for all biotopes investigated in this study. In hydrocarbon polluted soils, extremotolerant hydrocarbon degraders Rhodococcus ruber and R. erythropolis were found to be predominant (2.3x106-1.2x107 CFU g-1 of the total 2.2x107-4.1x108 CFU g-1 heterotrophs). For some isolates, the nucleotide sequences were identical with typical strains of new species R. qingshengii and R. wratislavensis. In heavy metal-contaminated soils, the 1.6-3.6-fold increase in the bacterial cell number, occurrence of metal resistant forms and 24-96% enhancement in dead plant biomass degradation was determined. Moreover, at low (Si=3-6) anthropogenic load, the bacterial cell number 11 -1 11 was as high as 1.5-2.4x10 CFU g , whereas at anthropogenic load of Si=23-30, it was 1.0-1.4x10 CFU g-1. This stimulation of bacterial communities was explained by the gradient increase in soil pH from 5.8 to 6.2-7.7 due to the presence of alkaline metals and evolution of metal-resistant bacterial forms. Our results confirm internal adaptation mechanisms of bacterial communities to xenobiotic contamination even under low temperatures but, on the other hand, they evidence the transformation of native ecosystems of cold climate to anthropogenic landscapes characterized by monospecies bacteriocenose formation, deep changes in bacterial species distribution and accumulation of resistant strains. The research was supported by Russian Federation Government (No. 11634.31.0036, 2010- 2011), RAS (No. 01201256869), Ural branch of RAS (No. 12-I-4-2051), and FP7-KBBE-2010-4 (BRIO No. 226106) grants.

129B Drying and rewetting events affecting the fate of sulfadiazine in rhizosphere soil and its effects on sul gene abundance Sven Jechalke*1, Ingrid Rosendahl2, Joost Groeneweg3, Jan Siemens2, Viviane Radl4, Holger Heuer1, Kornelia Smalla1 1Julius Kühn-Institut-Federal Research Centre for Cultivated Plants (JKI), Germany, 2Institute of Crop Science and Resource Conservation, Soil Science and Soil Ecology, University of Bonn, Germany, 3Institute of Bio- and Geosciences, Agrosphere, Forschungszentrum Jülich GmbH, Germany, 4Helmholtz Zentrum München, German Research Center for Environmental Health, Germany

Sulfadiazine (SDZ) is a veterinary antibiotic frequently used in the European Union. Due to its high excretion rates and persistence in manure it can reach agricultural soils where it can increase the risk of spreading antibiotic resistance. The fate and effects of organic compounds in soil may be influenced by microbial degradation, sorption or sequestration, which can influence their bioaccessibility. Changing soil moisture conditions such as naturally occurring drying-rewetting events may influence the microbial community structure and function as well as the dissipation of organic compounds in soil. However, only little knowledge is available so far of the role of soil moisture regarding dynamics and effects of antibiotics which would be of high concern for a reliable risk assessment.

In the present study, the influence of drying-rewetting cycles on the fate and effect of SDZ in the rhizosphere of grass was investigated. Soil microcosms planted with grass were treated with manure with and without addition of SDZ. These two soil treatments were incubated in climate chambers for 56 days at constant temperature. Water was added either regularly (control soil) to maintain moisture conditions of about 20-30% water content (water mass/soil dry weight) or in parallel was reduced in two drying events of 7 days resulting in a reduction of water contents down to 3%, each followed by a recovery phase of 20 days at about 20-30% water content (dry-wet soil). SDZ fractions of increasing binding strength serving as an approximation of the bioavailable concentration were obtained by a sequential SDZ-extraction protocol. The abundance and dynamics of sul1 and sul2 resistance genes relative to 16S rRNA gene copies were assessed by quantitative real-time PCR.

Slightly increased easily extractable SDZ concentrations were observed in dry-wet soil compared to the soil maintaining a constant moisture while concentrations in the residual fraction were not 32

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August significantly different suggesting a reduced (bio-)transformation or slower sequestration in the dry-wet soil. In accordance with the results of previous soil microcosm experiments, manure containing SDZ had a significant effect on sul1 and sul2 relative abundance (Anova, P < 0.05) which was mostly increased in the SDZ-treatment. Drying-rewetting cycles significantly influenced sul2 relative abundance as a lower relative abundance of sul2 was observed after drying cycles for the treatment without SDZ, while for the SDZ treatment the abundance of sul2 was in the same range as in the control soil. The observed results suggest a small but significant effect of drying and rewetting cycles on bacterial populations carrying sul2 resistance genes leading to a decrease in relative abundance in soil. This decrease was less pronounced in the presence of SDZ indicating a selective advantage compensating for the drying and rewetting effects. The increase in easily extractable SDZ concentration in dry-wet soil compared to the control probably was too small to obtain detectable effects on sul relative abundance.

130B Towards defining bio-indicateurs of arsenic mobility in soils Catherine Joulian*, Jennifer Harris-Hellal, Anne Mercier, Valerie Laperche, Francis Garrido, Fabienne Battaglia-Brunet French geological survey (BRGM), France

Arsenic (As) pollution in soils is a major health and environmental threat to water resources. Arsenic bioavailability in the environment is directly influenced by communities of As(III) oxidizing and As(V) reducing bacteria that catalyse the transformation of As(III) to As(V), a form easily precipitated, and the transformation of As(V) into As(III), the more mobile and toxic form, respectively. The objective of our work is to define molecular bio-indicators of arsenic mobility, in parallel with existing physic- chemical methods, and to evaluate their ability to predict As behaviour in the environment.

Incubations of two soil samples from an industrial waste land containing different amounts of As (about 500 and 1600 ppm) showed that in aerobic conditions, favorable to As(III) oxidation, the microbial community stabilizes the arsenic by maintaining it as As(V). In anaerobic conditions, with the addition of a source of exogenic carbon to stimulate heterotrophic bacteria, the decrease in redox potential induces conditions in favor of As(V) reduction: 70% of the initial As(V) is solubilized. Without carbon addition, As solubilisation and As(V) reduction occur simultaneously in conditions where the redox potential is not favorable for As(V) respiration. The different As(III) oxidizing and As(V) reducing- bacterial communities issue from these incubations were studied, aiming in particular an As(III)- oxidase gene, aioB and an As(V)-reductase, coded by arrA. The pattern of these genes was also investigated in a set of 10 soil samples from the same waste land, covering a broader range of total As content (100 to 10000 ppm). Overall, a link between the evolution of these functional genes (quantity, diversity, ratio) and content, speciation and behavior of inorganic As in soil will be discussed.

131B Diversity and abundance of nitrous oxide reductase genes associated with Bacteroidetes in organic compound-rich soils Jaejoon Jung*1, Jaemin Noh1, Sungjong Choi1, Hoon Jung1, Kate Scow2, Woojun Park1 1Korea University, South Korea, 2University of California, Davis, United States

The nosZ gene encodes nitrous oxide reductase, a key enzyme in nitrous oxide reduction during denitrification. Many conventional approaches have used proteobacteria-based primers for detecting nosZ in environmental soils. However, these primers often fail to detect nosZ from non-proteobacteria strains, including Firmicutes (Gram-positive) and Bacteroidetes. In contrast, newly-designed nosZ primers successfully amplified this gene from five Geobacillus species (Firmicutes). These primers were used to construct nosZ clone libraries from DNA extracted from sludge and domestic animal feedlot soils, all of which contained high organic carbon contents. Phylogenetic analysis have identified many new nosZ sequences showing high levels of homology to nosZ from Bacteroidetes, probably because of the high sequence similarity of nosZ from Firmicutes and Bacteroidetes, and a predominance of Bacteroidetes in feedlot environments. Quantitative real-time PCR with another three newly designed primers based on clone library sequences have revealed that a significant amount of Bacteroidetes nosZ existed in soil environments. The specificity of new primers was confirmed by new clone library construction and sequencing. Conventional primers would not have detected many of the nosZ sequences in soil. Thus, it appears that amplification with conventional primers is insufficient for developing an understanding of the diversity and abundance of nosZ genes in the environment. 33

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132B Effect of aeration and high efficiency microorganisms in controlling emissions and pathogen outbreak from mortality disposal system: a bench-scale study of in-vessel stabilization of carcass digestion Md. ANirban Jyoti*1, Sang-Seob Lee1, Weon-Wha Jeong2, John J. Bang3 1Kyonggi University, South Korea, 2Environmental Infrastructure Research Department Water Supply & Sewerage Research Division, National institute of Environmental Research, South Korea, 3Department of Environmental, Earth and Geospatial Science, North Carolina Central University, USA

Recent outbreaks of foot and mouth disease (FMD) caused deaths of several animals in South Korea. Protective measures like sacrificing infected animals added the numbers of dead animals in the region. To encounter environmental safety-guards from such animal mortalities safe disposal is necessary to control malodor emissions and possible pathogen outbreak. Therefore, in this bench- scale study we built an in-vessel carcass disposal system and applied high efficiency microorganisms in order to control emissions and increase the biodegradability.

Control (C), aerobic (A) anaerobic (An) vessels were prepared in duplicate by adding same volume of soil (10kg) and pork meat (2.5 kg). In control no high efficiency microbial inoculums were added. On the other hand, 1 gram/kg soil (wet weight) of High Efficiency Microbial inoculums (designated as KEM) was prepared and applied in aerobic (A) and anaerobic (An) vessels. In case of aerobic vessels (A), aeration was supplied by an aerated pump. Gas samples were collected from each vessel periodically until week 30 and analyzed by chromatography. Additionally, weights and photographs were also recorded periodically. Cotton-swabbing of soil and decomposed carcasses of each vessel were collected periodically and subjected for microbial community structure and diversity analysis by denaturing gradient gel electrophoresis (DGGE).

In this study, during the 30 weeks of degradation period aerobic vessels (A) lost a total of 0.029% weights, whereas in anaerobic (An) and control (C) vessels weight loss was recorded as 0.025% and 0.023% respectively showing the order of degradation in-vessel as A>An>C. Both aerobic and anaerobic degradation caused CO2 emission in the vessels Ammonification due to microbial degradation of in-vessel carcass was observed through measuring ammonia concentration periodically until week 30. The highest peak of ammonia was observed at week 6 in control (C=25489pm), aerobic (A=23311ppm) and anaerobic vessels (38398pm). From week 8, a slower ammonification was observed and ammonia stabilized in week 20. Trimethyl amine was observed to emit high at week 8 in control (C=1.8%), Aerobic (A=1.117%) and anaerobic (An=1.7%). In-vessel amine emission rate was observed in the order of C>An>A. However, emission of TMA was stabilized from week 20 in all vessels. Sulfur related compounds appeared mostly during week 6 to 20 in all vessels. However, at week 6, methyl mercaptan was found in aerobic vessel (65076ppm) at higher concentrations than the control (46128ppm) and anaerobic vessels (60079ppm). The order of sulfur gas stabilization in all vessels was found higher in aerobic conditions following the order of A>An>C.Microbial community analysis through DGGE bands showed distinct phylogenetic divergence between control, aerobic and anaerobic vessels over time. However, DGGE bands corresponding to applied KEM was shown to stable in aerobic and anaerobic vessels.

These results showed that application of both aerated oxygen and high efficiency microorganisms was beneficial for stabilization of carcass disposal system by decreasing the concentrations of emissions, shortening the degradation time and increasing the safe-guards.

133B Soil fungal communities and enzyme activities in a sandy, highly weathered tropical soil treated with biochemically contrasting organic residues Bhanudacha Kamolmanit1, Patma Vityakon1, Wanwipa Kaewpradit1, Georg Cadisch2, Frank Rasche*2 1Khon Kaen University/Department of Plant Science and Agricultural Resources, Thailand, 2University of Hohenheim/Institute of Plant Production and Agroecology in the Tropics and Subtropics, Germany

Biochemical composition of organic residues, whose quality is partly defined by their content of N (Norg), and polyphenols (PP), influences soil microbial communities and associated decomposition 34

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August processes. To provide complementary information on how organic residue quality influences soil microbial properties in tropical soils, we used a long-term field experiment on a highly weathered sandy soil in Northeast Thailand in which effects of biochemically different organic residues including -1 -1 rice straw (RS; Norg content: 4.7 g kg ; PP content: 6.5 g kg ), groundnut (GN; 22.8; 12.9), tamarind (TM; 13.6; 31.5), and dipterocarp (DP; 5.7; 64.9)) on soil organic matter dynamics are being studied for 16 years. Our interest was how particularly fungal communities and enzyme activities in the soils receiving these residues for 16 years (“native” soils, N-soil) were altered as compared to those that did not receive any residue material during the same period (control soil; C-soil). Soils were taken and subjected to a baseline study providing information on how 16 years residue application has altered the fungal community in N-soils as assayed by 18S rRNA gene-based community profiling using T- RFLP and sequence analysis. To elucidate long-term effects on specific microbial processes, another soil proportion was incubated for 56 days with the following treatments: N-soils (NRS, NGN, NDP, and NTM) mixed with either residue (NRS+RS, NGN+GN, NDP+DP, NTM+TM) or left untreated, and C- soil mixed with either residue (C+RS, C+GN, C+DP, and C+TM) or left untreated (CON). Incubated soils were analyzed for activities of invertase, beta-glucosidase, phenoloxidase and peroxidase. Results from the baseline study revealed distinct structural differences of the fungal community as induced by long-term addition of contrasting organic residues. All soils, in particular CON and NRS, were dominated by Myceliopthora sp. Residue quality related differences were further revealed by Aspergillus sp as predominantly found in NGN and NDP soils. Majority of clones in NTM soils were affiliated with Cryptococcus sp. and Fusarium sp. Results from the incubation showed highest phenoloxidase and peroxidase activity in C+DP and NGN soils, while activities of invertase and beta- glucosidase were promoted in NTM+TM and NGN+GN soils. Results indicated that 16 years of continuous residue addition induced a distinct change in soil fungal communities and promoted microbial processes in N-soils suggesting the stimulating effect of organic residues. The most obvious residue quality effect was shown in microbial phenoloxidase activity in C+DP soils, probably because of the high polyphenol content in DP. Eventhough fungal communities were different under NGN and NTM soils compared to CON, they showed similar patterns of phenoloxidase activity. We proved that long-term addition of different residues shaped soil microbial communities and processes, but it remains unsolved why C-soils showed similar enzymatic responses to N-soils and why polyphenol oxidation was pronounced. To answer this, we will identify those fungal community members involved in polyphenol oxidation using functional gene studies.

134B Response of soil microbial community against pulse perturbation by aromatic pollutants Hiromi Kato*1, Hiroshi Mori2, Fumito Maruyama3, Atsushi Toyoda4, Yoshiyuki Ohtsubo1, Genki Fuchu1, Ryo Endo1, Ayumi Dozono2, Masatoshi Miyakoshi5, Yuji Nagata1, Asao Fujiyama6, Ken Kurokawa2, Masataka Tsuda1 1Tohoku University, Japan, 2Tokyo Institute of Technology, Japan, 3Tokyo Medical and Dental University, Japan, 4National Institute of Genetics, Japan, 5University of Würzburg, Germany, 6National Institute of Informatics, Japan

To know the flexibility of soil microbial community and its gene pool against chemical perturbation, we conducted sequential metagenomic analysis of closed system of soil that was artificially contaminated by aromatic pollutants. Four aromatic compounds simultaneously dissolved in methanol were adsorbed on silica powder, and they were, after the removal of methanol, added to the experimental soil. Metagenome samples prepared periodically from the contaminated and control soils were used for the pyrosequencing of PCR amplicons of 16S rRNA genes as well as for the Illumina GA IIx- sequencing. The ten-million Illumina short reads were subjected to the homology searches against databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG) and A CLAssification of Mobile genetic Elements (ACLAME). The microbial response in the control soil, where a trace amount of methanol remained, was simple; transient increase of methanotrophs was observed, and the community structure rapidly returned to the original one. In contrast, much complicated microbial response was observed in the contaminated soil; transient domination of various taxa such as Burkholderia, Dyella, Sphingomonas and Acidobacterium occurred at different time points, and the community structure returned slowly, but not completely, to the original one at least 12 weeks after the disappearance of pollutants. During this community succession in the contaminated soil, abundance of genes for various metabolic pathways also changed and tended to return to the original state. Dynamic patterns of gene abundance of metabolic pathways during the experimental period were clustered into three groups: (i) the group responding drastically to the degradation event of the 35

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August pollutants (pathways for xenobiotic degradation), (ii) the group responding moderately to the event (for example pathways for xenobiotic degradation, several amino acid and carbohydrate metabolism, cell membrane metabolism, and etc), and (iii) the group remaining constant (many essential pathways). Our detailed analysis of the genes for aerobic degradation pathways of the pollutants suggested the involvement of multiple microbial taxa in the sequential reactions for degradation of the pollutants although the initial oxygenation reactions were inferred to be mediated by a few taxa (for example Mycobacterium). A number of reads assigned to phage genes were found only in the metagenome samples from the contaminated soil, and the prophages from Burkholderia were inferred to be induced by reactive oxygen species that could be generated during the aerobic oxygenation reactions of pollutants. In conclusion, our sequential metagenomic analysis allowed to characterize the fluctuation patterns of gene abundance of various metabolic pathways, and is also informative for the system- level understanding of the robust community of soil microbes.

135B The soil metaproteome of two different soil samples - a comparison of protein extraction protocols Katharina Maria Keiblinger*1, Ines C Wilhartitz2, Thomas Schneider3, Leo Eberl3, Katharina Riedel4, Sophie Zechmeister-Boltenstern5 1University of Natural Resources and Life Sciences, Austria, 2Eawag, Department of Environmental Microbiology, Switzerland, 3University of Zurich, Institute of Plant Biology, Switzerland, 4Institute of Microbiology, University of Greifswald, Germany, 5Institute for Soil Research, Department of Forest and Soil Sciences, University of Natural Resources and Life Sciences (BOKU), Austria

Metaproteomics and its potential applications are very promising to study microbial activity in environmental samples and to obtain a deeper understanding of microbial interactions. However, the complexity of soil samples constitutes a major challenge in extracting proteins from this matrix. A spiking experiment was conducted to evaluate protein recovery from soil samples. Sterilized forest soil was amended with proteins from pure cultures of Pectobacterium carotovorum and Aspergillus nidulans and analyzed with two different extraction methods. The protein recovery in the spiking experiment was almost 50%. Moreover, we compared soil protein extraction protocols in terms of their protein extraction efficiency for two different soil types, a potting soil and a forest soil, which are both high in clay content. Four different protein extraction procedures were applied based on (a) SDS extraction without phenol, (b) NaOH and subsequent phenol extraction, (c) SDS – phenol extraction and (d) SDS – phenol extraction with prior washing steps. This approach enabled us to compare the protein yield for the respective protocols. To assess the suitability of these methods for the functional analysis of the soil metaproteome proteins were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) and the number of unique spectra and assigned proteins for each of the respective protocols was compared. In order to assign proteins to phylogenetic and functional groups a comprehensive database including metagenomic and meta- transcriptomics sequence information and a bioinformatic pipeline allowing semi-automatic data evaluation were used. Our results show that the choice of the extraction protocol used for a certain soil sample can affect yields of proteins and to some extend influence the obtained diversity pattern. In both soil types, extraction with SDS-Phenol (c) resulted in “high” numbers of proteins. The function of “amino acid transport and metabolism” for example for all protocols higher in the potting soil compared to the forest soil. The observed overlap of unique spectra for all four different protocols was very low with 0.9% and 2.9% for potting soil and forest soil, respectively. Relatively low numbers of proteins (ranging from 80-494) were extracted from the soil matrix. Therefore we assume that every protocol has unique biases towards certain sets of proteins. Overall, our study demonstrates that a critical evaluation of the extraction protocol is crucial for the quality of the metaproteomics data, especially in highly complex samples like natural soils.

136B Controls on the function and genetic diversity of the coupled processes of nitrification and denitrification in tributary sediments Haryun Kim*1, A. Ogram2, K. R. Reddy2, H. Bae2 1BioEMCo, CNRS, France, 2University of Florida, United States

Denitrification is largely dependent on nitrification for supply of nitrate; however, nitrification and denitrification are typically controlled by different environmental factors and do not co-vary in many 36

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August environments. Denitrification is an important mechanism for nitrogen removal from small streams, such that the insight into the fundamental factors controlling the two processes is important for prediction of nitrogen removal rates. Our research investigated the relationships between biogeochemical properties, potential rates, and genetic structures and abundances related to archaeal amoA (AOA) and nirS responsible for nitrification and denitrification, respectively in tributary sediments of Santa Fe River in North Florida, USA. Potential nitrification rates and abundances of AOA were significantly and positively correlated with potential denitrification rates and abundances of nirS, respectively in tributary sediments. The pH was positively correlated with both potential nitrification rates and the structure distributions of AOA in our samples. The diversity of AOA was negatively correlated with carbon and nitrogen contents of sediments, while the abundance of AOA was positively related to carbon and nitrogen contents, probably due to the over growth of few species under high carbon and nitrogen conditions. Potential denitrification rates were regulated by the TC:TN ratios in riparian sediments, suggesting the importance of carbon quality to their functions, while the abundances and structure distributions of nirS correlated with carbon and nitrogen quantities in tributary sediments. The distributions of AOA and nirS genotypes co-varied, which might be due to the inter-correlation between organic carbon and pH. Thus, the coupling of community structures and abundances suggests that the potential nitrification and denitrification would be tightly linked in tributary sediments. In summary, our studies showed the importance of coupled nitrification and denitrification processes, and the relationships between biogeochemical factors, functions and structures of AOA and nirS in tributary sediments.

137B Metatranscriptome analysis of microbial community in flooded rice microcosm Yongkyu Kim*, Werner Liesack Max Planck Institute for terrestrial microbiology, Germany

CH4 is the second most important greenhouse gas with a global warming potential about 25 times greater than CO2. Flooded rice fields are one of the major biogenic sources of atmospheric methane. The emission of CH4 is the net balance between CH4 production by methanogenic archaea in anoxic bulk soil and CH4 oxidation by methanotrophic bacteria in the oxic surface layer and rice rhizosphere. In this study, we analyzed and compared the metatranscriptome of microbial communities inhabiting the oxic surface layer and anoxic bulk soil of flooded rice paddy soil microcosms incubated in the greenhouse under normal day/night cycles. Samples for metranscriptome analysis were taken from three different time points, corresponding to the different growth stages of the rice plant: tillering, flowering, and ripening.

Our metatranscriptome pipeline comprised extraction of total RNA or enriched mRNA, cDNA library construction, 454 GS-FLX Titanium pyrosequencing, and bioinformatic data analysis. The pyrosequencing reads obtained from total RNA were assigned to rRNA or non-rRNA by comparison against the SILVA rRNA database. SSU rRNA sequences (ribo-tags) were used to determine the taxonomic composition of the microbial communities in the oxic and anoxic zones of the flooded microcosms. A total of 10,000 SSU rRNA sequences were obtained for each zone and time point by this PCR-independent approach. In addition, we analyzed 45,000 and 12,000 putative mRNA sequences from the oxic and anoxic zone, respectively, of 90-day-old microcosms (ripening stage).

No major temporal changes in the composition of the microbial communities were observed, except for clostridia and methanogens in the anoxic zone. The percentage of ribo-tags assigned to these two groups increased from 8 % to 13.5 % and 1.5 % to 5.1 %, respectively, over time. However, the taxonomic composition of microbial communities were clearly distinct between samples from the oxic and anoxic zone (percentages of ribo-tags): Cyanobacteria (40-55% versus 2-4%), methanotrophs (4% versus 2.8%), Anaeromyxobacter (1.3% versus 5.5%), Geobacter (0.5% versus 5%), actinobacteria (2.7% versus 12%), clostridia (Crenothrix-like bacteria were prominent in the oxic zone, while type II methanotrophs (Methylocystis) were detected in the anoxic zone. Most abundant methanogens were members of the Methanosarcinaceae and Methanocellales.

The proportion of putative mRNA sequences that could be functionally annotated was clearly higher in samples from the oxic zone (60%) than in those from the anoxic zone (40%), suggesting that the anaerobic microbial community is less well represented by public genome databases. The functional annotation patterns of mRNA transcripts showed distinct differences in relation to light and oxygen 37

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August availability. For example, transcripts encoding proteins of photosynthesis and pyruvate metabolism I (anaplerotic reactions) were characteristic of the oxic surface layer, while those encoding proteins of methanogenesis and pyruvate metabolism II (acetyl-CoA, acetogenesis from pyruvate) were representative of the anoxic zone.

Our research provides a mean to analyze total composition and functional gene expression of soil microbial communities while avoiding the limitations of PCR-based approaches.

138B Methanol consumption by methylotrophs is correlated with vegetation type, pH, and substrate concentration in aerated soils Steffen Kolb*, Astrid Stacheter, Harold L. Drake University of Bayreuth, Germany

Methanol is the second most abundant organic molecule in the atmosphere. Its main source are methoxy groups of plant polymers (such as pectin and lignin). Aerobic methanol oxidation in soils might be an important sink in the global methanol cycle, and have previously not been addressed at in situ relevant methanol concentrations. In addition, few studies have analysed structures of non- methanotrophic methylotrophic communities in soils.

Apparent Michaelis-Menten kinetics of material of two aerated and temperate grassland soils were determined, which were supplemented with 14C-methanol. 14C carbon dioxide production was measured, and recovery of 14C was calculated. Localisation of methanol oxidation activity ‘hot spots’ was analysed in grassland soil by supplementation of 14C methanol. Soil slurries with supplemental cyanide served as controls for abiotic activity, and were not substantially active compared to cyanide- free treatments. Community structures of aerobic methylotrophs were assessed by amplicon pyrosequencing and TRFLP of structural genes of the monocarbon metabolism, that is mxaF (methanol dehydrogenase), mch (methenyl cyclohydrolase), and fae (formaldehyde activating enzyme).

Cyanide-treated samples did not reveal substantial methanol oxidation rates compared with untreated samples. Washed roots from a grassland soil and sterile Arabidopsis thaliana plants exhibited lower methanol oxidation rates than root-free soil. Thus, soil organisms were the main drivers of methanol oxidation. mch-TRFLP analysis of the methylotrophic community indicated that two alphaproteobacterial genotypes (GL1 and GL2) responded to low (that is micromolar) methanol concentrations. GL1 and GL2 were closely related to mch of Hyphomicrobium denitrificans that harbours a high affinity methanol dehydrogenase with an apparent Michaelis-Menten constant at the micromolar concentration range. In six grassland and six forest soils, environmental factors were identified that influenced the methylotroph community structure based on mxaF, mch and fae. Thus, vegetation type (forest or grassland), soil pH, the availability of nitrogen and substrate concentration likely determine which methylotroph taxa are involved in the flux of methanol from aerated soils of temperate ecosystems.

139B Organic matter decomposition: feedbacks on plant and microbial community structure Jan Kopecky*, Veronika Hrdinkova, Marketa Sagova-Mareckova Crop Research Institute, Czech Republic

Microbial community is responsible for degradation of organic matter, the quantity and quality of which is dependent on the plant community. There must be a link determining the microbial community structure as a response to the available organic matter. Above that, the organic matter is enriched with plant secondary metabolites which may be complemented with the secondary metabolites of the microorganisms acting in accord in structuring the microbial community. Within the soil microbial community, actinobacteria fulfill distinctive ecosystem functions through their diversified metabolic activities. They are highly efficient in degrading complex plant polymers and interact heavily with their soil chemical environment, which provides the substrates for their metabolic processes and at the same time is modified through metabolic activities of actinobacteria. However, little is known about the significance of actinobacterial metabolite production for ecosystem processes. C and N cycling during decomposition are controlled by the content and diversity of chemical compounds within plant litter

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PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August mixtures. Therefore, the diversity of chemical compounds in litter is a potentially important functional trait affecting decomposition.

The project aimed at establishing specific relationships between composition of plant litter and its decomposers. In particular, the effect of a specific site and its locally selected microbial community was compared to the effect of plant litter quality. We hypothesized that plant litter would decompose at a different rates according to its chemical composition and that composition of low molecular weight compounds including secondary metabolites would be correlated to both plant litter quality and site.

A field litterbag experiment has been established at two contrasting locations. A meadow site, temperate steppe with prevailing forbs and a beech forest were chosen based on previous screening. As substrates, three plants differing in chemical composition were used. The litterbags and surrounding soil were sampled in two-month intervals. From the sampled litterbags, residual weights were determined and aliquots of the remaining litter were subjected to DNA extraction and analysis to determine microbial community structure, or extracted with organic solvents and analyzed by HPLC- UV for the profiles of low molecular weight compounds, both of plant origin and decomposition products.

In accordance with expectations, decomposition rate seemed to differ among the three substrates. The observed decomposition rates showed large differences depending on litterbag position at the site, i.e. high local variability of decomposition processes. It was in agreement with previous studies on enzymatic activities in forest soil, which described occurrence of active decomposition centers with higher activities of enzymes acting in decomposition at the spots correlated with microbial activity. The consequences of plant chemical diversity and abiotic soil characteristics on the decomposition processes are discussed.

140B Metabolic potential of pesticide-contaminated soils revealed with deep sequencing and metagenomic analysis Pawel Krawczyk*1, Marcin Golebiewski2, Marcin Ostajewski2, Dorota Tokarska1, Michal Kaminski3, Adamska Dorota1, Marta Blaszkiewicz3, Adam Sobczak1, Leszek Lipinski1, Andrzej Dziembowski1 1Institute of Biochemistry and Biophysics PAS, Poland, 2Department of Biotechnology, Nicolaus Copernicus University, Poland, 3Institute of Genetics and Biotechnology, University of Warsaw, Poland

Pesticides are heterogeneous group of chemical compounds causing one of the most acute environmental problems, both in land and aquatic ecosystems. Many pesticides, usually of organochlorine origin, are very stable and recalcitrant compounds accumulating in the food chain and causing adverse effects on human health and the environment. Soil is the habitat which is the most affected by peciticides, mainly due to modern extensive agriculture. At the same time soil is one of the richest habitats in terms of bacterial diversity and source of many important compounds of bacterial origin like small molecules, antibiotics or enzymes. What is more, high evolution rates of bacteria allow them to quickly adapt to changing environmental conditions and upon chemical contamination develop novel enzymes and metabolic pathways.

Those facts trigerred us to address the question how prolonged exposure to pesticides influence bacterial communities and their metabolic potential. We analyzed soil microbial communities from samples collected in close proximity to expired pesticide burial areas in Poland, where hundreds tons of toxic compunds where stored for more than 30 years. DNA was isolated with modified protocol of Zhou et al (1996) and bacterial diversity was estimated by 16S rDNA amplicon sequencing. Metagenomic DNA from selected samples was sequenced using Illumina technology, then assembled and annotated using developed pipeline.

The analyses showed negative correlation between level of pesticides contamination and species richness. Additionally, when high concentrations of chlorinated compounds were present in soil, we observed increase in Gamma-Proteobacteria content and species from Pseudomonadaceae genus were the major group of organisms found by 16S rDNA analysis.

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To better understand observed correlation we performed a deep sequencing of selected samples. Assembly of 300Gb of sequencing data yielded about 5 million open reading frames which were annotated against protein sequence (NCBI nr, PFAM, TIGRFAM, COG) and metabolic pathways databases (KEGG, SEED). Analysis performed with MEGAN software revealed that over 11% of potential ORFs in metagenoms from highly contaminated soils might be involved in xenobiotics degradation in comparison to only 6-7% in less polluted samples. During a deeper analysis we have also found an increase in the content of proteins annotated as probable dehalogenases in organochlorine-rich samples.

Presented results show that contamination of soil with chlorinated compounds cause severe perturbations in species content and their metabolic potential. Obtained data indicate pesticide- polluted soils as an interesting object for studying adaptation of bacterial metabolism to persistent chemical compounds present in the environment.

141B Effects of quality and quantity of plant-derived organic substrates on the fungal communities present in an arable soil Dirk Krüger*, Julia Moll, Björn Hoppe, Kezia Goldmann, François Buscot Helmholtz Centre for Environmental Research - UFZ, Germany

As soil microorganisms, fungi play an important role in nutrient cycles, because one of their chief functions lies in decomposing organic matter. In the frame of the DFG- (German Research Foundation) funded research unit FOR 918 „Carbon flow in belowground food webs assessed by isotope tracers“ we investigate the role of saprobiotic fungi in the transfer of organic carbon from plant origin to belowground food webs of an agricultural soil. To tackle how carbon quality and availability influence the fungal communities, a field experiment has been installed where two crops, maize (Zea mays L.) und wheat (Triticum aestivum L.), are cultivated in a design with and without the addition of maize litter. Soil from three depths was sampled in July, September and December in 2009 and 2010 to analyze seasonal shifts in the fungal community composition. F-ARISA (fungal automated ribosomal intergenic spacer analysis) resulted in 198 OTUs (operational taxonomic units). Univariate statistical analysis revealed that this fungal species richness correlates with crop species and litter addition. The fungal species richness was highest in September in both years. Multivariate statistical analysis demonstrated that the soil fungal community is mostly affected by soil depth, followed by the impact of the plants and related root exudates. These results indicate strong reactions of the fungi to different nutrient supplies. In a microcosm series with different nutrients the fungal key players actively assimilating carbon were identified using rRNA-SIP (stable isotope probing) and T-RFLP. In follow-up studies, the taxonomic affiliation of these fungi must be sought.

142B Phytase producing bacteria from Himalayan soil and their tricalcium phosphate solubilizing abilities Vinod Kumar*1, Prashant Singh1, Punesh Sangwan2, A. K. Verma1, Sanjeev Agrawal1 1G.B. Pant University of Agriculture and Technology, Pantnagar, India, 2C.C.S. Haryana Agriculture University, Hisar, India

Phosphate solubilizing bacteria (PSB) are involved in mineral phosphate solubilization process through secretion of organic acids, phytases and phosphatases. Phytases are enzymes which catalyze the hydrolysis of phosphomonoester bonds in phytate, thereby releasing lower forms of myo- inositol phosphates and inorganic phosphate contributing to plant nutrition. In the present study, seven bacterial isolates from Himalayan soil with potential phytase activity were chosen to evaluate their tricalcium phosphate solubilization potential to select potential microbial inoculants for the agricultural purposes. Bacterial identification was done using 16S rDNA gene sequencing and GenBank accession no. has been assigned to submitted sequences for all isolates. Bacterial Strain PB-13 has shown maximal phytase activity (0.2390 U/ml), whereas highest tricalcium phosphate solubilization was achieved by strain PB-09 (746.8 µg/ml). Maximum solubilization index of 4 was obtained by strain PB-06 on Pikovskaya agar plates. On the basis of 16S rDNA, isolates were found to be in two clusters but no genetic relationship could be established between isolates on the basis of phytase activity, pH and phosphate solubilization potential. Use of these PSB as bioinoculants might increase the available phosphorus (P) in soil, helps to reduce the P-fertilizer application, minimizes environmental P pollution and promotes sustainable agriculture. 40

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143B Characterization of Microbial communitites in sub-surface flow constructed wetlands of United Arab Emirates Rajkumari Kumaraswamy*, Rahsed Algaoud, Jorge Rodríguez Masdar Institute of Science and Technology, United Arab Emirates

Treatment of domestic wastewater using constructed wetlands is a rapidly emerging technology in the areas not suitable for conventional treatments and also less expensive. The sub-surface flow constructed wetlands (SSFCW) has very high scope and utility in United Arab Emirates (UAE). Currently SSFCW in UAE are being operated using the guidelines from other regions and very few studies have been conducted about their designs, operations and implementations.

The efficiency of constructed wetlands is mainly driven by the microbial reactions such as COD removal, nitrification, denitirification, nitrogen fixation etc. Therefore, it is very important to study the microbial communities of different constructed wetlands in UAE and reflecting with their performances. So far, no study has been conducted for microbial characterization which will enable the end users to have a better control over the operations of wetlands and also optimize their performances.

In the current study solid samples from SSFCWs from different locations in the UAE are collected for microbial community studies. In each location, sub-surface soil microorganisms at different reed bed depths are characterised in the context of the specific SSFCWs characteristics, history and performance. In addition, specific sampling campaigns are conducted in newly started SSFCWs where microbial samples are taken at different depths and times after initial sewage feeding start-up. This aims at revealing the microbial community development and shifts over time and depth during the initial weeks of the CW operation under local conditions.

Two different but complementary molecular tools are used to acquire the identity & abundance of microorganisms and the microbial population shifts. Random 16S rRNA sequencing of the soil DNA samples (pyro-sequencing) can be conducted which reveal the identity and abundance of different microbial groups. A rapid molecular biology tool PCR-DGGE of 16S rRNA gene is used complementary to study the population shifts. The results of the above experiments can reveal the differences in the microbial composition, identity and abundance relative to location, depth of the sub- surface soil and time points after initial sewage feeding. The results obtained will be the first of such nature in constructed wetland studies for the UAE.

144B Combined impacts of climate changes and anthropogenic activities on dryland soil microbiota of the Southwest United States Cheryl Kuske*1, Blaire Steven1, Chris Yeager1, Jayne Belnap2, Sasha Reed2 1Los Alamos National Laboratory, USA, 2United States Geological Survey, USA

Dryland ecosystems constitute about 40% of the Earth’s landscape. Sparse plant cover is characteristic of dryland ecosystems, and plant interspaces can comprise up to 70% of the ground surface. Biological soil crusts (BSCs), comprised of photosynthetic and diazotrophic cyanobacteria, other bacteria, fungi, lichens and mosses, colonize the interspace soils. Thus, both the plants with their associated soil microbiota and the BSCs are important contributors to C and N inputs and soil stability in dryland ecosystems. Dryland ecosystems are acutely sensitive to environmental perturbations. The combined effects of predicted changes in climate patterns and increased anthropogenic activity in drylands globally, are expected to impact these sensitive ecosystems, but the extent of impact, potential interactive effects and their potential to recover remain unknown. The long term goals of our studies are to (1) assess the combined impacts of a variety of co-occurring stresses, including increased soil temperature, altered precipitation patterns, elevated atmospheric CO2, N- deposition, and physical disturbance, on the soil bacterial and fungal communities inhabiting plant root zones and BSCs; and (2) determine the potential for maintenance of their biological and ecological functions as conditions in dryland ecosystems change.

We are using a variety of nucleic-acid-based approaches to assess bacterial and fungal community biomass, relative abundance and community composition in multiple large-scale manipulated field experiments located in the Colorado Plateau and Mojave regions of the Southwest USA. Combining quantitative PCR with targeted and shotgun metagenomic approaches, we are focusing on soil 41

PS16 – Soil Microbiology and Heterogeneity Tuesday 21 August bacteria and fungi that are important structural components of the BSCs and that are instrumental in cycling C and N in BSCs and plant root zones.

An altered summer precipitation pattern had rapid (within 2 yrs), negative impacts on the cyanobacteria and mosses in BSCs in a dryland grassland. Soil temperature increases of 2-3°C also resulted in BSC decline, but only after 5 yrs of treatment. The response to ten years of elevated atmospheric CO2 was more subtle, resulting in a measurable reduction in biomass of BSC cyanobacteria, as well as alterations in shrub-associated bacterial and fungal root zone community structure. Once-a-year foot stamping of surface soils over a decade resulted in loss of biological soil crust structure, reduced soil N, and increased erosion.

An altered summer precipitation pattern and chronic physical disturbance dramatically and negatively impacted the surface soil microbial communities. Soil warming and elevated atmospheric CO2 conditions resulted in more subtle direct effects on the soil communities, but contribute important feedbacks to atmospheric climate that are predicted to result in altered precipitation patterns for this region. The resilience to physical disturbance and potential for continued C and N inputs by soil communities under altered climate regimes is being monitored, and the links between soil microbial community C and N cycling in plant root zones and the BSCs are being explored.

145B Cell abundance and microbial community composition over a complete oil sand mining and recultivation process Michael Lappé*, Beate Schneider, Jens Kallmeyer University of Potsdam / Institute of Earth and Environmental Science, Germany

Hydrocarbons constitute an important carbon source for microbes. Microbial activity causes hydrocarbon alteration over geologic timescales and is therefore of economic relevance.

The present study characterizes the abundance of microbes along the oil sand mining process in Alberta, Canada, as a first approach to assess the impact the mining and extraction process has on the microbial population of the substrates involved.

The oil is extracted from the sediment by hot-water extraction (50-60°C), resu