Genomics:GTL

Environmental Remediation Challenge continued

The Role of Microbial Systems in Remediation

Microbes found in the contaminated subsurface and other environments often have the metabolic capability to degrade or otherwise transform contaminants of concern to DOE. Currently, DOE environmental restoration is targeting soluble forms of toxic metals and radionuclides in soils and sediments that move through the groundwater. Subsurface microbes, through their interactions with each other and the geochemical environment, play a role in modifying the geochemistry of these subsurface environments, thereby affecting their chemical form and movement. Microbes can directly, or indirectly through their influence on sediment geochemistry, provide a potential cost-effective bioremediation strategy to immobilize contaminants. Shewanella and Geobacter, for example, are two types of microbes that can enzymatically transform toxic species such as Uranium(VI), which is soluble and moves in groundwater, to Uranium(IV), which is insoluble and precipitates as UO2 (uraninite) (see sidebar, BER Research Advancing the Science of Bioremediation).

Benefits and Impacts

Although comparisons of the cost and effectiveness of metal and radionuclide bioremediation strategies with those of traditional remediation methods are not available, the cost savings for bioremediation of organics are estimated to range from 30 to 90%. In addition, in situ bioremediation, taking advantage of natural microbial populations in the subsurface, has the potential to reduce costs and increase the efficiency of groundwater treatment as compared to conventional pump-and-treat technology. Given that over 1 billion m³ of water and 55 million m³ of solid media at DOE sites in 29 states are contaminated with radionuclides (Linking Legacies 1997), potential savings accrued by use of innovative technologies are likely to amount to billions of dollars (Bioventing 1996; Patrinos 2005; Scott 1998).

Research is needed to provide useful information to decision makers on whether remediation is necessary and practical, give an accurate prediction of contaminant mobility, and suggest bioremediation strategies. A biotreatment technique that works well at one site may perform poorly at another because we lack understanding of the unique interactions—in these “geologically powered dark ecosystems”—between the microbial community and subsurface geochemistry (Nealson 2005). Characterization and monitoring tools must be developed to gain that understanding. At a few sites of specific interest to DOE, less than 1% of the microorganisms have been collected, cultured, and characterized in any great detail, and only a small fraction of those have had their genomes sequenced. Even less is known regarding the interactions of microorganisms in communities. We have only begun to appreciate the existence of such systems, let alone understand them so we can take advantage of their diverse capabilities (Gold 1992).

Establishing the Link Between Biology and Geochemistry

A key to successfully understanding these systems will lie in establishing the link between biology and geochemistry. An exacerbating challenge in establishing a meaningful science base for future applications is the inherent complexity of the subsurface and the distribution of contaminants. Most contaminated sites have extremely heterogeneous geology, hydrology, and resultant geochemistry. Subsurface processes are difficult to measure, control, and monitor, in part because samples from monitoring wells provide only single timepoint data from a large three-dimensional area and because monitoring wells can disturb subtle interactions.

Contaminants do not flow uniformly from a source point and can be transformed physically, chemically, or biologically to alter their state and mobility. Microbial and geochemical processes can, for example, both immobilize contaminants through redox (oxidation/reduction) processes and enhance transport through complexation. These natural and induced heterogeneities dramatically affect the distribution and makeup of microbial communities, resulting in countless niche environments and communities that must be understood and for which remediation strategies must account.

Using Genome Sequences as a Launch Point to Understand Communities

In this complex venue, we first must define the genomic potential of microbial communities. GTL uses genome sequences as a launch point for detailed, mechanistic investigations into microbial metabolism and other cellular subsystems to achieve a systems-level understanding. Having complete genome sequences for microbes known to catalyze important contaminant-transformation reactions provides an unprecedented opportunity to describe these processes, from molecules through systems. Genome sequences enable investigations of the identities and functions of individual genes that code for important contaminant-transformation reactions, and their expression can be related to field-scale models of transformation processes in the environment—an important advance from earlier, more qualitative descriptions. Whereas historically our studies have been limited to microbes that can be cultured in the laboratory, the combination of metagenomics with the production and characterization of proteins from genes allows new insights into microbial function.

Most current research focuses on understanding single microbial species having potential for environmental remediation and stabilization (e.g., Shewanella, Desulfovibrio, and Geobacter) in laboratory-based cultivation or field studies. Microbially mediated environmental processes, however, rarely are due to the activity of a single group of organisms—microbes, even those in contaminated environments, typically live in diverse communities. Little is known of the overall dynamics of these communities, and the complexity and spatial structure of energy-transfer reactions that occur across the microbe-mineral interface have only begun to be revealed (see Research Highlights). Characterization of microbial communities generally has been investigated using single-gene (16S rRNA) sequencing surveys to gain phylogenetic insights. Only a few organisms (notably metal and sulfate reducers) have been sequenced and characterized to any extent. Obtaining detailed links between genome sequence and molecular mechanistic function will require the most robust metagenomic techniques to assess the makeup of communities and their genomic potential.

Modeling Microbial Metabolic Activities

Mechanistic models of microbial communities are key requirements in constructing field-scale contaminant fate and transport models that extrapolate over very long time periods. These models must treat such aspects of microbial metabolism as reactions to growth, stress, and nutrient limitation (among many others) that can directly affect gene expression. Understanding changes in all these metabolic activities along the migration pathway or with respect to time requires mathematical models. These models accurately simulate microbial metabolism and are supported by data from biogeochemical and environmental measurements to viably reflect the dynamic interplay of microbes and environment (see sidebar, A Revolutionary Whole-Genome Perspective). Results will form the basis for evaluating and modeling pathways of such cellular processes as signaling, regulation, and response to contaminants. Key elements of modeling scenarios include microbe-mineral interactions and resulting molecular structural and charge-transfer responses; microbial-community responses (e.g., signaling, motility, biofilm formation, and other structural responses); and ensuing community functionality.

Merging Metabolic and Field-Scale Models

Eventually, GTL in silico genome-based microbial-metabolism models must be merged with Environmental Remediation Sciences Division (ERSD) field-scale models of contaminant fate and transport. Some of these techniques already are being generated within environmental-restoration programs and GTL. GTL currently is focusing on quantitatively deciphering the molecular and biochemical pathways of several model microbes that catalyze contaminant-transformation reactions of interest to DOE. Additionally, complementary research within ERSD focuses on understanding the biogeochemical potential of subsurface microbes. [For more information, see Bioremediation of Metals and Radionuclides: What it is and How it Works; 2^nd ed., 2003.] To accomplish this linkage, more trained scientists are needed to determine the makeup of subsurface microbial communities and their interactions with the geochemical environment.

GTL’s Vision for Environmental Remediation and Restoration

GTL science will facilitate detailed, large-scale discovery and investigation of microbes and microbial ecosystems with important contaminant-transformation capabilities. These studies will expand knowledge about structure, function, metabolic activity, and the dynamic nature of microbial communities and their interaction with the geochemical environment. The information will aid in the prediction of microbe-mediated contaminant fate and transport by providing more reliable, science-based information upon which to base remediation decisions. Capabilities and information established by GTL in conjunction with ERSD programs also will enable the integration of predictive microbiology with remediation science, leading to more effective and reliable applications.

Gaps in Scientific Understanding

Details underlying successful field-scale models ultimately focus on dynamic microbe and microbial-system geochemical interactions and functionality but have their foundations in the molecular interactions and processes that GTL seeks to understand. The following outlines, in broad terms, the science required over the next 15 years to deliver an integrated physiology and genomics-based understanding of microbial metabolism to support the detailed modeling of microbially catalyzed contaminant transformation in the environment. Key questions that we must be able to answer include the following:

• What is the makeup of microbial communities? We need to learn who is there, their physiological states, their individual contributions, how they relate to each other and the environment, and how metagenomic DNA sequence can be used to predict the function, behavior, and evolutionary trajectory of microbial communities.
• How do microbes identify, access, and modify their local geochemical environments to gain energy and nutrients and meet other metabolic requirements?
• What physicochemical environmental interactions control the dynamic makeup, structure, and function of microbial communities in the subsurface, and what is the resultant impact on contaminant transformation?
• How and why do contaminants impact microbial communities, and what are potential indicators of these impacts?
• How do molecular mechanistic processes in microbes and communities relate to macroscopic behaviors in field environments?

Bioremediation Challenges, Scale, and Complexity

Research and Analytical Challenges

Scale and Complexity

Analysis of microbial communities and their metabolic activities that impact the fate and transport of contaminants Analysis of geochemical changes in subsurface environments due to microbial or chemical activity Accurate conceptual and quantitative models for coupling and scaling microbial processes to complex heterogeneous environments

Hundreds of different sites, millions of genes, thousands of unique species and functions Functional analysis of potentially thousands of enzymes involved in microbe-mineral interactions; hundreds of regulatory processes and interactions; spatially resolved community formation, structure, and function; influence on contaminant fate Models at the molecular, cellular, and community levels incorporating signaling, sensing, metabolism, transport, biofilm, cell-mineral interactions; incorporated into macromodels for fate and transport

Defining Microbial Communities and Their Potential

• Metagenomic methods tailored to unique subsurface communities and environments to assess their general and specific makeup. These studies will identify biochemical potential where possible and determine unknown genes through protein production and characterization and other methods. Improving high-throughput sequencing is allowing recovery of the genetic potential of single-strain microbes and whole communities (Venter et al. 2004; Tyson et al. 2004).
• Refinement of the growing genome sequence databases and bioinformatics tools to identify and analyze genes found in environmental organisms. New and faster methods of genome annotation (“super annotation”) are required to capture fully the genetic potential of sequenced organisms.
• Modeling and experimental capabilities to explore the physiology of full systems. Current genomic descriptions of microbial metabolism for sequenced species are based on genes and the proteins they encode of known function or inferred from similar gene sequences found in other organisms such as E. coli. Genes of unknown function, which constitute a significant portion of most sequenced genomes, cannot be modeled. We must be able to produce and characterize these unknown proteins on demand. The sheer number of novel gene sequences to be investigated dwarfs currently available investigative techniques (such as knockout mutant characterization).
• Genome annotation to include functional genomics information such as cell response to environmental stimuli using functional gene arrays or expressed protein analyses. These results will develop the basis for identifying and modeling biochemical pathways and regulatory networks within cells, including growth, stress, and metabolic responses to potential contaminants and other environmental factors.
• Additional genome sequence generated for microbes and microbial communities and increased computing power to enable more-intricate comparative genomics studies. Also, more extensive protein structural analysis, protein network analysis, and fold-recognition applications will be possible.
• Method refinements to monitor growth and activities of microbial communities within the subsurface. GTL will develop and standardize technologies for extraction of mRNA and protein from environmental samples. Information provided by these analyses forms crucial links between results obtained from current GTL research and the microbial processes as they occur in the environment.
• Improved cultivation methods such as the microdroplet technique (Zengler et al. 2002; Keller and Zengler 2004) and others to capture a greater proportion of microbes associated with environmental samples. These capabilities will enable us to study samples in the laboratory and compare them to the more thoroughly studied model organisms.

Measuring Microbial Processes and Responses

Breakthrough capabilities and technologies are needed to support measuring and modeling of microbially mediated contaminant-transformation processes and microbe-mineral interactions. These tools should permit a more complete mechanistic understanding of microbially mediated processes within the environment and provide solutions for issues related to microbiological and geochemical scaling needed for field-scale descriptions. Capabilities will be developed for modeling and measuring communities and single cells, both in isolation and within communities, to understand microbial systems. Examples include the following:

• New, sensitive methods to measure the proteome, metabolome, and transcriptome of populations and communities of organisms. Such measurements will involve significant developments in the area of high-throughput gene sequencing, protein identification and production, multivariable cultivation techniques, controlled cultivation and physiological analyses, and enzymatic analysis.
• Methods to determine the biochemical basis for intracellular and intercellular interactions that contribute to the functionality of biofilms and other structured communities.
• Multifunctional imaging techniques to monitor biological, chemical, and physical changes simultaneously within environmental samples.
• Methods to investigate subsurface biogeochemical processes. These require further development because most remain focused on either the geochemical or biological aspects of subsurface processes, to better integrate mechanistic aspects of both. As examples,
  • Several microspectroscopic techniques afford detailed analyses of mineral and biological composition and structure at high resolution and small scales and can be further developed for use with live biological analyses.
  • Kinetic data gathered from nutrient-enriched cultures grown in the lab often do not reflect processes observed in the field. Computer models of contaminant fate and transport within the environment must be developed to include robust biogeochemical modules. New techniques and capabilities are needed to apply laboratory data to complex field environments.
• Techniques to examine and identify the composition of natural organic matter for exploring the metabolism of naturally derived substrates in environmental samples. Only a subset of this material currently can be identified.
• Data to characterize natural communities, including the following:
  • Microbe-microbe interactions such as cell signaling, materials and energy transfer, gene transfer, and syntrophy to study phenomena occurring in biofilms. They remain poorly understood at a mechanistic level.
  • Functional microbial-community analysis to understand microbial positioning, including the potential for biofilm and other structured community development; their structure and relationship to function; and the molecular basis for microbial motility, competition, and niche exploitation. Little is known about the molecular basis for changes in community structure resulting from shifts in environmental conditions such as the introduction of contaminants. Detailed information about the impact of environmental changes on microbial-community structure leads to understanding of the community's functional stability and the net metabolic flux of electron donors and acceptors, including contaminant transformation. This knowledge will lead to better, more environmentally relevant descriptions of microbe-mediated processes.

Microbe-Mineral Interactions

Techniques will be developed for evaluating electron transfer and growth at mineral surfaces. Many microbes of interest to DOE respire metals (i.e., iron and manganese) that typically exist in solid-phase minerals. These organisms oxidize organic compounds and hydrogen and ultimately pass these electrons on to mineral surfaces, capturing energy for growth during the process. A crucial component of investigation is the ability to image and quantify processes occurring at the microbe-mineral interface, including the following:

• The molecular basis for changes in cell-membrane structure to understand attachment, electron transfer, and mineral chemistry at the microbe-mineral interface. More detailed analyses at the microbe-mineral interface will include molecular-level analyses of cell-membrane composition and dynamics and the interactions among membrane proteins and potential contaminants and mineral phases that can serve as electron acceptors.
• Methods to measure microbial-community and environmental chemical and physical structures using methods that combine, for example, nuclear magnetic resonance (NMR) and optical imaging technologies.
• Further development of tomographic and spectromicroscopic techniques to provide information about chemical changes occurring on and, to a certain extent, within mineral surfaces, as well as about metals associated with microbial cells.
• Application of established and novel microscopies in combination with new sample-preparation and spectroscopic techniques to observe bacteria attaching to and growing on mineral surfaces in a noninvasive, real-time fashion. These new techniques, in addition to infrared and micro-NMR imaging, will be needed to evaluate microbe-mineral associations under noninvasive conditions and in real time, a significant challenge.
• Imaging abilities coupled with probes designed to evaluate metabolic processes as they occur within cells and communities of cells at a mineral surface in real time. Coupled visual measurements of biological and geochemical parameters at the cellular scale will aid development of temporal and structural descriptions of microbial communities at mineral interfaces.
• New suites of biosensors or probes to measure a variety of metabolic and geochemical processes down to the microbe-mineral scale. These tools must be specific enough to relate biochemical changes to geochemical processes at high spatial resolution and extremely small scale. The sensors, along with required advances in cultivation techniques, will permit the investigation of microbially mediated processes, not only under controlled laboratory conditions but also within the environment. Once these processes can be measured and evaluated, new methods of kinetics analysis will be required for accurate testing and evaluation.

Modeling and Simulation Capabilities and Data Management

• Novel analysis techniques to mate the results of GTL in silico models of microbial metabolism with field-scale contaminant fate and transport models created within DOE ERSD. This is part of a classic problem in relating the temporal, spatial, and process scales of molecular mechanisms to the scales of macrosystems such as contaminant plumes that function over years and kilometers.
• Computational techniques to correlate spatial information with geochemical properties and reactions at meaningful scales.
• Improved modeling techniques to enable incorporation of many more variables, particularly when attempting to simulate molecular processes at larger scales or under a variety of environmental conditions. Current modeling capabilities and analysis tools are geared to mathematical representations that are too simple or too small (<10-state variables).
• Significant improvements in data management and interpretation to use and distribute the enormous amounts of data generated by genome sequencing and associated systems biology. Current bioinformatic techniques are limited primarily to sequence and structure analyses of genetic information. The vast amount of biological and environmental data presents equally pressing challenges for storing and managing access to these very large data sets. Organization is key, and centralization is envisioned in which input of and access to data can be standardized to common QA/QC protocols and routines. Metadata definitions and databases must be formalized and managed in a similar fashion.
• Significant computer power and efficient computational methods to support visualization techniques, particularly important for protein structure and 3D simulation of processes in both the laboratory and environment.

Text adapted from Genomics:GTL Roadmap: Systems Biology for Energy and Environment, U.S. Department of Energy Office of Science, August 2005. DOE/SC-0090.