The ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area (SFA) 
is a national laboratory and university consortium managed by Lawrence Berkeley National Laboratory that seeks to advance the increasingly high resolution mapping from molecules to microbes and leverage the discovery of mechanisms composing the subcellular, cellular, and intercellular networks of metabolite, protein, RNA and DNA molecules that drive macroscopic biogeochemical processes, integrating these into the larger framework of interacting microbial communities and ecosystems to address DOE goals.
The Oak Ridge National Laboratory team within ENIGMA is currently part of the Community Dynamics & Environmental Transformation and the Microbial Physiology, Networks & Structural Organization groups while working closely with the Environmental Technologies group. The main goal of the ORNL team is to develop and utilize enriched microbial consortia and isolates from the field as well as constructed model consortia to support building a predictive, systems-level understanding of complex microbial communities. This will be achieved in a step wise fashion through hypothesis driven research and in collaboration with different ENIGMA groups. More specifically, we aim to characterize the structure, function and metabolic interplay of individual populations within microbial communities, their contributions to the resulting biogeochemistry and the effect of contaminating physicochemical pressures on the community. Secondly, establish enriched consortia from the field using controlled cultivation and elucidate population succession through composition and function using meta-systems biology tools. We propose to identify, isolate and characterize “keystone” microorganisms using systems biology tools and bring them to model organism status. Keystone microbes will be used to create model microbial communities so that conditions can be controlled to reveal the mechanisms of interspecies relationships (syntrophy /competition) and to ultimately predictive models of complex microbial systems in collaboration with other ENIGMA researchers. Finally, these studies will supply ENIGMA with the highest quality samples and datasets in order to support high resolution and high-throughput analyses towards the ultimate goal of developing predictive models of microbial activity and microbially driven changes in biogeochemistry.
Statement of Core Interest and Expertise. The ORNL team core interest is to identify and understand the presence, method and mode of communication between the dissimilar microorganisms that constitute a microbial community. This communication (syntrophic or competitive), forms the structure & function of keystone community members while permitting transient microorganisms to join the community. Further, we believe that it is this community makeup and its’ stability over time that constitutes the resultant biogeochemical changes observed and the potential for large scale subsurface immobilization of metals of interest to DOE. The expertise of the ORNL team is diverse. Dr. Elias is a DOE recognized expert in microbial physiology and bioremediation, Drs. Podar and Brown are DOE recognized experts in meta- and functional- genomics, and Drs. Phelps and Palumbo are internationally recognized experts in microbial physiology and community ecology, respectively.
Community Dynamics & Environmental Transformation
- Characterize the structure and function of the overall microbial community as well as those of the individual populations within the community.
- Determine which populations of the “core” microbial community are successful under environmental pressures present at contaminated DOE field sites; i.e. enriched consortia.
- Characterize and derive the relative biogeochemical contributions of the enriched consortia members using meta-systems biology tools including meta- genomics, transcriptomics, proteomics.
- Supply all groups within ENIGMA with the highest quality samples and datasets in order to support high resolution and high throughput platforms to achieve predictive models of microbial communities at the most discreet levels.
Natural microbial community experiments. The experiments conducted with the natural community from Hanford will serve several functions. For the Community Dynamics team, the experiments will serve as
1) a multi-year baseline or foundation of the community structure & function to indicate how stable the in-situ and successive, enriched community is from the same area.
2) reveal which in-situ and enriched populations are tolerant to varying [Cr(VI)].
3) establish the similarity & dissimilarity of in-situ and enriched communities along the Cr(VI) gradient.
4) determine the in-situ and enriched community response to varying co-contaminants.
For the Microbial Physiology team, attainment of enriched, less diverse consortia and new field isolates allows for more discreet experiments, including bringing isolates to model organism status and developing genetic manipulation systems to answer key questions.
Other ENIGMA groups will also benefit including the technology groups to
1) identify and characterize proteinaceous and non- proteinaceous signaling molecules from individual isolates.
2) enhance technologies such as the Geochip and Phylochip to include sequences that may currently be missed. This is particularly relevant if keystone organisms are not currently accounted for by these latter technologies. In each natural community experiment, we will coordinate our planktonic conditions with M. Fields (Montana State University) to compare with biofilm community succession.
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| Temporal bacterial community composition from 16S rDNA pyrosequencing analysis of selected dates from a triplicate continuous flow reactor experiment of lactate-enriched Hanford well H-100 groundwater sample. Over time, the genus Pelosinus became a dominant member of the community. | Metal-reduction assays from four separate strains of Pelosinus isolated from Hanford 100H along with the type strain Pelosinus fermentans, showing the chromium and uranium reduction capability of these new isolates. |
Microbial Physiology, Networks & Structural Organization
- Create and characterize model microbial communities that are relevant to the DOE mission using systems biology tools so as to reveal interspecies relationships of syntrophy or competition.
- Isolate, identify, and characterize new species that are keystone members of the community using physiological, metabolic, and systems biology tools (genomics, functional genomics, transcriptomics, proteomics).
Model constructed microbial community experiments. The model microbial community experiments previously performed (eg. Miller et al., 2010) and those in the present and near future serve two primary purposes.
1) To be able to perform, analyze and achieve a deeper, more discreet understanding of the physiological and metabolic interactions between bacteria since they are predetermined, model organisms. This allows for the employment of omics tools that can be mapped to discreet genes.
2) To close the lab to field gap by incorporating new isolated species from the field over the FY2012-FY2014 period as they begin to attain model organism status. Such is the case for the new Hanford Pelosinus strains as below.
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| Proposed model of the metabolic flux and carbon and electron balance of the three member community of Clostridium cellulolyticum (C.c.), Desulfovibrio vulgaris (DvH) and Geobacter sulfurreducens (G.s.). * Values given are in moles. ** Circled electron equivalents could be hydrogen, interspecies electron transfer, or ethanol. From Miller et al., 2010. BMC Microbiology. 10:149 |
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| (A) Growth and temporal organic acid profiles for the chemostatic, syntrophic growth of D. vulgaris and G. sulfurreducens using lactate as the carbon and electron source and fumarate as the electron-acceptor for G. sulfurreducens. D. vulgaris had no electron-acceptor and relied on G. sulfurreducens to maintain a low H2 partial pressure while G. sulfurreducens relied on D. vulgaris for acetate (from the oxidation of lactate) as its’ carbon and electron source. | (B) Species specific fluorescent monoclonal antibodies (a gift from T. Hazen) for D. vulgaris (red) and G. sulfurreducens (blue) showing the population proportions and close proximity of the two species. |
Environmental Technologies
- Develop new analytical tools and techniques such as simultaneous DNA and RNA isolation from various subsurface matrices including clays high in iron and humic acids.
- Isolate, identify, and characterize new species that are keystone members of the community using physiological, metabolic, and systems biology tools (genomics, functional genomics, transcriptomics, proteomics).
