Subsurface Biosphere Initiative Workshop/ IGERT Retreat
June 18-21, 2006

Abstracts of Poster Presentations
Monday, June 19, 2006

In Author Order

The Contributions of Bacteria and Fungi to Nitrogen Cycling in Forest Soils under Douglas fir and Red Alder

Stephanie A. Boyle, Department of Crop and Soil Science, Peter J. Bottomley, Department of Crop and Soil Science and Department of Microbiology, David D. Myrold, Department of Microbiology, Oregon State University

Previous studies have found that in soils with high N concentrations gross N cycling rates increase, nitrate (NO3-) accumulates, and N losses from soil are greater. Presumably these differences result from changes in the functioning of soil microbes, but knowledge of the links between microbial community composition and N cycling rates remains limited. A study was conducted to better understand the relationship between N-cycling and the soil microbial community by examining net and gross N transformation rates in conjunction with microbial community structure under pure stands of red alder and Douglas fir at two sites in Oregon. A combination of 15N-tracer experiments and community profiles were used to investigate the relative abundance and roles of bacteria and fungi in N cycling under different tree types and at sites with high vs. low productivity. These experiments revealed that N cycles differently in low versus high N forest soils and the relative contributions of bacteria and fungi change in response to low and high productivity sites. Community compositional differences include a lower fungal: bacterial ratio and an increase in the fraction of Gram positive bacteria at a highly productive site. These observations suggest that in highly productive forest soils, bacteria may play a more significant role in the overall cycling of soil N and emphasize the importance of understanding soil microbial communities as a means to better understand biogeochemical processes.

Woody Plant Invasion of Grassland Alters the Composition of Soil Microbial Communities

Elizabeth A. Brewer, Department of Crop and Soil Science, Oregon State University, Thomas W. Boutton, Department of Rangeland Ecology and Management, Texas A&M University, David D. Myrold, Department of Crop and Soil Science, Oregon State University

Woody plant encroachment into grasslands alters the quantity and quality of litter and root inputs to soil, and often modifies the storage and dynamics of soil organic matter. These changes in the functional composition of plant communities and key ecosystem processes may have significant impacts on the structure and diversity of soil microbial communities. Our objective was to examine changes in soil microbial communities in the subtropical grasslands of southern Texas that have been invaded by trees and shrubs. Soils from two depths (0-15 and 15-30 cm) were collected at four positions along transects extending from the center of shrub/tree clusters out into the surrounding open grassland. Phospholipid ester-linked fatty acid (PLFA) analysis was used to characterize the composition of the soil microbial communities. PLFA analyses revealed significant differences in the composition of the microbial communities along these transects at the 0-15 cm depth, but not at 15-30 cm. For example, the ratio of fungal to bacterial PLFA’s decreased significantly from the center of the woody clusters to the open grassland. These results indicate that soil microbial communities are altered following woody plant encroachment into this subtropical grassland ecosystem.

Ecological Soil Community Management for Enhanced Nutrient Cycling in Organic Sweet Cherry Orchards

Lisa Brutcher, Anita Azarenko, Annie Chosinski, Russell Ingham, David Myrold, Clark Seavert, Oregon State University

Sweet cherry growers transitioning to organic practices face considerable barriers, including: higher labor, managerial, and certification costs; loss of production during the transitional period; risk associated with unfamiliar management practices; and, especially, a lack of information specific to the organic system. Organic sweet cherries still command a premium price, unlike organic apples and pears (ERS 2002, Hinman and Watson 2003), and have various environmental advantages. A 21-year study by Mader et al. 2002 comparing organic and conventional agricultural systems found that the organic systems supported higher soil microbial biomass and activity, had higher nutrient use efficiency, and required substantially less pesticide. Soil microbial biomass plays a central role in the efficient recycling of nutrients from organic litter and converting nutrients from organic to plant available, mineralized forms (Coleman and Crossley 1996). The composition, activity, and biomass of the soil communities is significantly influenced by orchard floor management practices (Forge et al. 2003, Neher 1999). Changes in soil biodiversity have also been associated with N mineralization and availability (Setala 2002, De Ruiter et al. 1993). To date only a few studies have examined the relationship between orchard management practices and soil microbial communities (e.g., Oved et al. 2001, Goh et al. 2001, Rutto 2002).

The goal of this research is to increase our basic understanding of the microbial contributions to nitrogen cycling dynamics in orchard systems - looking at the impacts of orchard floor management on soil microbial community structure and function, especially on AOB and denitrifying bacteria populations; and correlations between soil microbial community characteristics and nitrogen availability in orchard systems. The study sites are two first-year experimental plantings. Soil samples will be evaluated for activities of key nutrient cycling enzymes (protease, urease, laccase, arylamidase, NAG-ase, and beta-glucosidase); N-mineralization rates; quantification of nitrogen present in ammonia, nitrate, amino sugar-N and Uric acid-N forms; fungal to bacterial biomass ratio (by qPCR); and abundance (by qPCR) and community composition (by T-RFLP) of ammonia oxidizing bacteria and denitrifying bacteria.

Iron Reduction / Oxidation in Sulfurihydrogenibium spp. from Calcite Springs, a Terrestrial Hot Spring in Yellowstone National Park

Sara L. Caldwell, Anna-Louise Reysenbach, Department of Biology, Portland State University, Portland, OR

Examples of microbially-mediated iron reduction or oxidation are well documented under a wide variety of environmental conditions, but it is still unclear whether there are microorganisms capable of both iron reduction and oxidation. This investigation will use Sulfurihydrogenibium azorense, a recently-described member of the thermophilic group of bacteria, Aquificales, as a model to demonstrate iron reduction and oxidation in the same organism. S. azorense is the closest phylogenetic relative, based on 16S rRNA sequence similarity, to a population of black filaments found at Calcite Springs in Yellowstone National Park. These black filaments are hypothesized to reduce iron and, as a result, store solid iron-sulfide minerals in their cellular periplasmic spaces. As spring water temperature drops and conditions become more oxidizing downstream from the source, the cells may oxidize the ferrous iron stored in their periplasm for respiration and growth, demonstrating remarkable metabolic plasticity. Microbial iron cycling contributes significantly to biomineralization of organic matter in low sulfate or nitrate environments, and affects a wide range of other biogeochemical processes. Therefore, understanding the complexity of iron metabolism (and metabolism of other metals) in a single population will contribute significantly to knowledge of the microbial influence on low-oxygen energy fluxes, environmental quality, and the biogeochemical cycling of other major elements, such as carbon, oxygen, and sulfur. Understanding microbially-mediated iron cycling may also provide insight into the evolution of Earth's atmosphere or of life on other planets.

Resistance and Resilience of Soil Microbial Structure and Enzymatic Activities to Heat Shocks

Guilherme Montandon Chaer1, Marcelo Ferreira Fernandes, David Myrold, Department of Crop and Soil Science, and Peter Bottomley, Department of Microbiology, Oregon State University

In microbial systems, functional stability is not necessarily related to community stability. This is because there are many possible combinations and organizations of species that can produce similar ecological functions. To test this hypothesis in soil environments we artificially induced the disruption of the functional and community stability by exposing two soils (agricultural and forest sites) to increasing levels of heat-shocks (40, 50, 60 or 70oC for 15 minutes). Accordingly, we expected that a disturbance level would be reached that would led to a different microbial community structure that would still be able to carry out the same functions (levels of enzyme activity). Changes in the microbial structure were tracked by analyzing soil PLFA profiles at 3, 10 and 30 days after the heat-shocks. Concurrently, we analyzed the hydrolysis of fluorescein diacetate (FDA), and the activities of cellulase and laccase as indicators of the soil functional status. The analysis of the PLFA profiles revealed that the microbial community structure from the agricultural soil was more resistant to the heat-shock treatments than that from the forest soil. Moreover, the increasing levels of heat-shock in the forest soil suggested a threshold between 50 and 60oC that represents a limit for the expression of community structure resilience in this soil. The activities of all three soil enzymes decreased in proportion to the heat-shock level applied in both soils, but were generally resilient, especially in the forest soil. Despite the pronounced changes in microbial structure in the forest samples treated at 60 and 70oC, the enzymatic activities in these samples showed resilience over the 30 day incubation period after the heat-shocks. This observation supports the hypothesis of "ecological resilience" in soil environments, a situation where the new microbial structure established after a disturbance has the same functional capabilities than the previous one. However, our results also suggest that this phenomenon seems to be dependent on the intensity of the disturbance event.

Soil microbial and nematode community dynamics under elevated and depleted nitrogen conditions in paired sagebrush steppe and Bromus tectorum-invaded ecosystems

Nicole M. DeCrappeo, Forest and Rangeland Ecosystem Science Center, U.S. Geological Survey, and David A. Pyke, Department of Crop and Soil Science, Oregon State University

The invasion of Bromus tectorum (cheatgrass) into former Artemisia tridentata (big sagebrush) steppe communities in the Great Basin has resulted in a loss of plant species richness and structural diversity, changes in litter inputs, modification of the rhizosphere, and alteration of soil moisture and temperature regimes. These changes may have a significant effect on soil microbial and invertebrate community dynamics and consequent nutrient cycling. We characterized microbial and nematode abundance, composition, and functional diversity in paired sagebrush and cheatgrass plots in eastern Oregon and southern Idaho in October 2003 and June 2004. Carbon and nitrogen levels were experimentally manipulated using sucrose to test the hypothesis that cheatgrass production is suppressed at low soil N levels. Soil samples were collected directly under four cover types: sagebrush, bunchgrass, interspace (which included biological soil crusts), and cheatgrass. Soil microbial communities were assessed using both BIOLOG substrate utilization ecoplates and phospholipid fatty acid analysis (PLFA). Nematodes were extracted using a density-dependent sugar-centrifugation method. Principal components analysis (PCA) and nonmetric multidimensional scaling (NMS) were used to assess patterns in functional diversity and relative biomass for all taxa. We found that microbial substrate utilization was significantly different between cover types across sampling dates (p < 0.0001, A = 0.10 in October; p < 0.0001, A = 0.12 in June). Nematode communities also differed with cover type, with bacterial feeders dominating cheatgrass-invaded plots and root associates dominating under bunchgrasses. Carbon and nitrogen treatments had no measurable effect on microbial or nematode communities, suggesting that these organisms are more influenced by historical organic matter and rhizosphere inputs than ephemeral changes in nitrogen availability.

Expression of a Thermophilic Hydrogenase in Thermosynechococcus elongatus

Jed Eberly, Department of Biological and Ecological Engineering, Oregon State University

With current energy shortages a source of clean renewable energy must be developed. One potential solution to this problem is solar-based hydrogen production using cyanobacteria. Solar-based hydrogen production systems will generate heat, thus the effects of temperature on such systems is an important factor that needs to be addressed. In direct sunlight the absorption of infrared radiation could raise the temperature of a solar system to 50 C. One way to deal with the elevated temperature is by using thermophilic cyanobacteria. While hydrogen production has been studied in mesophilic cyanobacteria, to date no one has looked at hydrogen production by cyanobacteria at high temperatures. No thermophilic cyanobacteria are known to produce hydrogen. The goal of this project is to express a hydrogenase in Thermosynechococcus elongatus by transformation with a plasmid containing the hydrogenase genes from the thermophilic bacterium Acetomicrobium flavidium. T. elongatus is a thermophilic species of cyanobacteria found in hot springs at an optimum temperature of 55ºC. They are involved in photosynthesis driven sulfate reduction coupled with sulfate reducing bacteria and also play a role in the formation of stromatolites and sedimentary iron ores. T. elongatus has been completely sequenced and is naturally transformable which makes it an ideal model organism for studying hydrogen production at high temperatures. The poster discusses the background of the project and the procedures that will be used for expression of an active hydrogenase in T. elongatus.

Sinter Fabric Formation at K4 Well, Uzon Caldera, Kamchatka, RU: Implications for Biosignature Formation and Preservation

Jessica C. Goin and Sherry L. Cady, Portland State University

There are three types of biosignatures in the rock record that can provide evidence for early life. These are bona fide microfossils, chemical traces, and microbially influenced sedimentary structures (e.g. stromatolites). Many factors are involved in sinter fabric formation, and it can be difficult to determine biogenicity of a particular sinter fabric. In order to distinguish between abiogenic and biogenic sinter fabrics, and therefore increase the usefulness of this category of biosignatures, it is important to understand the role that the biofilm plays in the initial sinter formation. The relationship between biofacies and lithofacies at K4 Well provides an opportunity to study the impact that microbial communities have on the sinter fabric forming beneath them.

Interactions Between Soil Microbial Communities and Native and Non-native Invasive Plant Species After Wildfire in the Cascade Range of Oregon

Cassie L. Hebel, Department of Forest Science, Oregon State University, and Jane E. Smith, USDA Forest Service, Pacific Northwest Research Station

Suppression of wildfire for the last 100 years has led to large organic matter accumulations, contributing to high severity wildfires throughout the western United States. Small severely burned areas of soil associated with the consumption of large down wood or stumps in direct contact with the soil, are common after high severity wildfire. Excessive heating and oxidation of the soil matrix changes the top mineral layer to various shades of red. Such soils, largely void of biological activity immediately following a fire, are thought to increase the potential of invasion by non-native plants. We compared the soil microbial community and growth of 3 native and 3 non-native invasive plant species in paired samples of severely burned and less severely burned soils from the 2003 Booth and Bear Butte (B&B) wildfire, east of the Cascade Range of Oregon. Ordination results of phospholipid fatty acid (PLFA) analysis showed that soil microbes were most abundant in the less severely burned soils. Similarly, colonization by arbuscular mycorrhizal fungi (AMF) was greatest in plants grown in the less severely burned soils. Despite dramatic differences in AMF colonization, shoot biomass of several of the native and non-native invasive species did not differ between severely burned and less severely burned soils. Understanding the interactions among burn severity, soil microbial communities, and growth of native and non-native plants will assist forest managers with post-fire recovery.

Species Diversity of Mat Forming Ectomycorrhizal Fungi and Their Associated Microbial Activities

Laurel Kluber, Department of Crop and Soil Science, Susie Dunham, Department of Botany and Plant Pathology, Katie Tinnesand, Department of Microbiology, Peter Bottomley, Department of Crop and Soil Science and Department of Microbiology, Bruce Caldwell, Department of Forest Science, Kermit Cromack, Jr., Department of Forest Science, David Myrold, Department of Crop and Soil Science, Joey Spatafora, Department of Botany and Plant Pathology, Oregon State University

In 1999 we established an NSF-funded Microbial Observatory at the H.J. Andrews Experimental Forest. It was devoted to studying microbial community composition and activity in high-elevation meadows and forests. The current focus of our research at the Microbial Observatory is to examine microbial communities and activities associated with ectomycorrhizal (EcM) fungal mats. Through their activities, EcM mats can create a unique soil environment and likely result in the establishment of distinct microbial communities and activities. Our first research objective was to survey the phylogenetic diversity of EcM mat fungi in old-growth and second-growth stands dominated by Douglas-fir. Phylotyping by ITS amplification and sequencing found that species of Piloderma were the dominant mat-forming fungi in all stands, and that species of Ramaria and Hysterangium were of secondary importance in old-growth and second-growth stands, respectively. Our second objective was to survey soil biological and chemical properties of mat and non-mat soils in selected stands containing these dominant EcM phylotypes. Biological properties such as the activities of enzymes involved in processing organic C and N (e.g., N-acetyl glucosaminidase, ?- glucosidase, urease, and protease) were assayed. Soil characteristics, such as percent organic matter, water content, and pH were also measured. The poster will present and discuss the results of both research objectives.

Viral Mineralization in a High-Silicate Environment

James Laidler and Kenneth Stedman, Department of Biology, Portland State University

The discovery of the first microbial fossils by Walcott in 1914 and subsequent finds in later decades have had a tremendous impact on both biology and paleontology. Although controversy continues to surround some of these fossils, the existence of microbial life as far back as the Archaean period is gaining wider acceptance. One of the significant contributions of the early microbial fossils was to provide researchers with an idea of what microbial fossils might look like. This undoubtedly led to further discoveries of microbial fossils. The current lack of viral fossils can be attributed to two factors: [1] few, if any, people are looking for them and [2] there is no information on what viral fossils might look like. Many people looked at the same sort of carbonaceous deposits in the Gunflint Chert formation before Tyler and Barghoorn realized they were cyanobacteria. Likewise, paleontologists and geologists may have repeatedly looked at electron microscope views of viral fossils and not recognized them for what they are. The purpose of this project is to determine if viruses can be mineralized and to give some idea what fossilized viruses might look like. Since microbial mineralization has been observed in contemporary hot springs as well as in ancient hot spring relics, I will attempt to mineralize viruses under conditions similar to a silica-rich hot spring. The project will be carried out in three phases. The first phase will be to mineralize a range of viruses in the laboratory and examine them under the electron microscope. Once the mineralized viruses have been characterized, the second phase will be to look for the same characteristics in silica sinter from high-silica hot springs. If mineralized viruses can be unambiguously identified in contemporary hot spring silica deposits, the third phase will be to examine cherts that have fossilized microbes for the presence of fossilized viruses.

A Phylogenetic Review: Endolithic Microbes Associated with Marine Basalts

O. Mason, College of Oceanic and Atmospheric Sciences, Oregon State University, U. Stingl, Department of Microbiology, Oregon State University, C. Di Meo-Savoie, Medical University of South Carolina, Charleston, M. Fisk, College of Oceanic and Atmospheric Sciences, Oregon State University, and S. J. Giovannoni, Department of Microbiology, Oregon State University

Over the past decade textural, chemical and genetic evidence of endolithic microbes associated with marine basalts has been provided by several workers. Additionally, culture-dependent methods have recently been used to determine the metabolic status of these endoliths, and in some instances, to quantitatively determine the role of microbes in basalt glass dissolution. These cultivation based approaches have led to the successful isolation of metal-oxidizing microbes by several investigators. In addition, sequences from clone libraries of enrichment cultures where basalt glass was used as the innoculum have recently been generated (Mason, unpublished). The culture-dependent approaches discussed above have produced approximately fifty 16S rDNA sequences. Culture-independent approaches, where DNA was extracted directly from basalts, have also been employed. These molecularly based analyses have produced approximately 200 sequences. Taken together there are currently 240 sequences publicly available, and several unpublished sequences, that represent ten different Bacterial phyla and two Archaeal phyla. These sequences were elicited from basalts of varying ages collected from diverse geographic locations. This study seeks to examine the distribution of these endoliths to determine whether they are cosmopolitan in their distribution or endemic to certain geological environments. To address this question a database of sequences from basalt microbes has been established and phylogenetic trees generated to summarize the evolutionary relationships among these endoliths. The end product of this study will be a phylogenetic review that seeks to provide a common language with which to discuss the microbes associated with basalts and to examine their geographic distribution.

Comparison of Streamers and their Fossilized Counterparts in Silica-Depositing Hot Springs, Yellowstone National Park, USA

Hollie Oakes-Miller and Sherry L. Cady, Geomicrobiology and Electron Microscopy Laboratory, Department of Geology, Portland State University

Scanning electron microscopy (SEM) was used to address the question of what is the affect of silicification on the abundance and diversity of bacterial morphotypes in modern hot spring mat phototrophic streamer communities at various spatial scales. Three samples from the 55°C portion of the outflow apron of Queens Laundry hot spring were examined: unsilicified streamers, partially silicified streamers, and streamer sinter. The samples represent progressively more advanced levels of silicification. Two filamentous species, Chloroflexus and Phormidium, were found to be the dominant organisms in the two streamer samples. Several types of Synechococcus rods were also observed in the streamer samples, though they only made up a major fraction of the microbial population in the unsilicified sample. The filamentous morphotypes appeared most susceptible to silicification in all of the samples. In contrast, the Synechococcus species did not appear silicified in any of the samples. It is worth noting that the extracellular polysaccharides were silicified in the partially silicified streamer and streamer sinter, which lead to the preservation of the dominant architectural characteristics of the microbial streamer communities.

Moisture Effects on Respiration from Root-Associated Microbes

Claire L Phillips, Department of Forest Science, Oregon State University, Chris Andersen and Paul Rygiewicz, Western Ecology Division, Environmental Protection Agency, Barbara J. Bond, Department of Forest Science, Oregon State University

Root-associated microbes, including mycorrhizal fungi and free-living rhizosphere microbes, may contribute a significant proportion of total soil respiration in forest ecosystems. To assess the respiratory contribution from these types of microorganisms it is necessary to determine how they are impacted both by plant carbon supply and by soil conditions. We hypothesize that the component flux from root-associated microbes is greater under moist soil conditions than dry conditions, because under low soil moisture root-associate activity is negatively impacted by both reduced plant carbon supply and low moisture. We propose to test this hypothesis by determining respiration rates from root-associated microbes (Ra) at high and low moisture conditions in potted forest soil planted with 2-year-old Douglas-fir seedlings. The component flux Ra will be determined by the following equation:

Ra = Rt - Rr - Rh

where Rt is total soil respiration, Rr is root respiration, and Rh is heterotrophic respiration. Rr will measured from surface-sterilized roots removed from soil following measurement of Rt. Rh will be an estimate made of heterotrophic respiration derived from girdling a subset of trees to eliminate belowground transport of plant C. We anticipate that at high moisture levels, the sum of heterotrophic and root respiration will be less than total soil respiration, and that the difference is attributable to Ra. This difference is expected to be smaller under low moisture conditions due to the reduced contribution from root-associated microbes. To assess whether Ra is dependent on plant C, Ra will be standardized by root surface area and examined for correlation with canopy assimilation rates.

Genomic and metabolic variations between Nitrobacter winogradskyi Nb-255 and Nitrobacter hamburgensis X-14

Shawn. R. Starkenburg, Oregon State University, Patrick Chain, Joint Genome Institute and Lawrence Livermore National Laboratory, Luis Sayavedra-Soto, Oregon State University, Frank Larimer, Oak Ridge National Laboratory, Dan J. Arp and Peter J. Bottomley, Oregon State University

The Alphaproteobacteria Nitrobacter hamburgensis X14, participates in the process of nitrification by converting the end product of ammonia oxidation, nitrite (NO2-), into nitrate (NO3-). Sequencing and preliminary analysis of the N. hamburgensis genome was recently completed. Although N. hamburgensis is closely related to the previously sequenced N. winogradskyi (98% 16srRNA identity), genetic and phenotypic variations exist. The N. hamburgensis genome is approximately 1.4Mb larger than N. winogradskyi and consists of a main chromosome (4.4Mbp) and 3 plasmids. Many of the plasmid encoded genes consist of conjugal transfer/pilin formation subunits and heavy metal resistance proteins. The large plasmid, p27, contains a suite of genes important to central metabolism. Two copies of RuBisCO and the only copies of carboxysome encoding genes are present on p27. The heterotrophic capabilities of N. hamburgensis appear to be more robust than N. winogradskyi do to the presence of a complete glycolysis pathway and the presence of both formate and l-lactate dehydrogenases. The functionality of the l-lactate dehydrogenase was tested in both N. winogradskyi and N. hamburgensis. N. winogradskyi was not able to use lactate as a carbon or energy source. Conversely, lactate additions to N. hamburgensis cultures stimulated growth over 40%, even in the presence of excess CO2. Surprisingly, N. hamburgensis appears to use lactate strictly as a carbon source and not for energy. Further comparison of the two Nitrobacter genomes will provide new insight into the mechanisms which control nitrite oxidation and mixotrophy.