Real-Time Direct Environmental Assessment
The last decade has seen a revolution in information technology and in the power of microprocessors. It has also seen a quantum leap in the use of physical, chemical and biological sensors, as ever more sensitive detection techniques have been married to these powerful data processing engines. The field of environmental assessment has only recently awakened to the potential that these new sensors offer. It has also become painfully obvious over the past decade that the dynamics and true understanding of functional biogeochemical processes in all types of environments cannot be fully understood without direct physical, chemical, and biological measurements. Indeed, what goes on in the sampling device and sample bottle after collection may have nothing to do with the functional dynamics of the original environment that the sample was taken from, thereby effecting our fundamental understanding of that environment and how to characterize, monitor and control it. Hence, real-time direct environmental assessment is an area that the Center for Environmental Biotechnology (CEB), with its engineering, molecular biology, chemistry facilities, access to the Advanced Light Source and the Center for Isotope Geochemistry, can demonstrate unique R&D capability in this area. The Lawrence Berkeley National Laboratory has long provided scientific leadership in the disciplines of experimental physics, physical chemistry, geology, hydrology, geochemistry, molecular biology, modeling, and more recently natural attenuation and bioremediation technologies. Hence, CEB is well situated to capitalize on the Laboratorys strengths while advancing the science in this emerging field.
Real-Time Field Sampling and Monitoring
Ongoing projects within CEB are utilizing off-the-shelf flow and water quality sensors deployed within monitoring networks to help improve water management practices in both agricultural and wetland areas in the western San Joaquin Valley. These networks of sensors are also allowing scientists to obtain a greater understanding of selenium fate and transport within these areas and to forecast the environmental risk of selenium loading to the fragile Bay-Delta ecosystem. Selenium environmental risk assessment and bioremediation is an area in which the Laboratory has played an important leadership role in the past 15 years. An initiative has begun to develop a real-time sensor for selenium using the unique chemical properties of the metalloid as it is reduced from the mobile selenate species to the stable elemental form with its characteristic red coloration. Should this be accomplished, the potential savings to the State in reduced need for selenium analytical services could be measured in the tens of millions of dollars. Another new field application of these technologies will use real-time flow and water quality sensors, rapid laboratory assessment techniques and mathematical models to develop an early warning system for dissolved oxygen concentrations in the Stockton Deep Water Ship Channel. Low dissolved oxygen in the ship channel during certain critical times of the year act as a barrier to fish migration putting at risk Californias important salmon fishery an industry that the CALFED Bay-Delta Program will invest close to half a billion dollars to restore over the next decade. Two commercial manufacturers of field instruments have indicated an interest in working with CEB in the further development of these technologies. A spin-off from this research will be the study of carbon fluxes from managed seasonal wetlands in the San Joaquin Basin, an area that has been little studied and one that might have implications for future management of wetlands to sequester carbon. Carbon sequestration on a global scale has implications for future management of global warming, which is thought to be a factor in determining future climate.
Field sampling technologies
Applications of real-time monitoring techniques to the field often rely on the collection of soil, water or gas samples to obtain information on background environmental conditions. The collection of representative field samples of physical, chemical and biological materials is an often-overlooked aspect of monitoring. Although a great number of environmental sampling devices exist that purport to take accurate samples, many of these devices are crude and information on their performance is not available. This area of environmental monitoring is still in its infancy. CEB has benefited from the considerable field expertise developed over the years in the Kesterson Remediation Program, the Yucca Mountain Project and the EPA- sponsored Environmental Measurements Laboratory in the development of innovative field sampling techniques. Field collection of sediment samples in streams and canals is difficult and few devices exist that allow accurate discrete depth sampling. Researchers in CEB developed a simple portable sampling device for precise bed sediment sampling, which has been used successfully by agencies such as the US Bureau of Reclamation and Fish and Wildlife Service as well as UCB limnologists. Ongoing work within CEB continues to devise rapid, inexpensive techniques for environmental sampling.
Although commercial electronic sensors have improved both in cost and in accuracy over the past decade, certain chemical species continue to defy attempts for continuous field measurement. Microbial sensors have also been developed although this technology is still in its infancy. LBNL has a long-standing tradition for the development of innovative sensors, especially in the area of high-energy physics and physical chemistry. The materials science division continues to experiment with new materials with electrochemical and electrophysical properties that might be harnessed in new sensor technologies. Researchers within CEB have investigated microbial sensors for rapid assessment of selenium remediation potential and have initiated a collaboration with a researcher at the University of Maryland to develop a real-time selenium sensor using templated polymer technology. Ongoing collaborations in this area may explore combinations of electrochemical and biological properties of materials in the quest for novel environmental sensors. Electrochemical sensors are ubiquitous (pH, redox, oxygen, selective ion, etc). EETD collaborators in CEB have the capability to provide polymer membrane platforms for bio-and chemical sensors that connect with electrodes or spectroscopic sensing devices. Other departments in EETD have keen interest in the application of such sensors for air quality.Environmental modeling and decision support systemsAn emerging research area within CEB is the development of environmental modeling and decision support systems, which provide tools for the processing of real-time environmental data in a manner that assists managers making decisions. This cutting-edge area blends environmental systems analysis with the computer science fields of user interface design, information processing and data management. CEB has collaborated with researchers at UCB and within the Department of Water Resources and the US Bureau of Reclamation to develop environmental software for uses in real-time salinity management in the San Joaquin River and real-time salinity management in wetlands. Other projects have involved building graphical user interfaces to improve the efficiency of models used to predict groundwater conjunctive use, water allocation decisions and habitat development for wildfowl. Integration of models for environmental assessment is the subject of a funded collaboration with UC Berkeley, UC Davis, the Department of Water Resources and the Fish and Wildlife Service directed at assessing impacts in the San Joaquin Basin due to future climate change. Developing systems that integrate real-time environmental data acquisition, data processing, visualization and real-time decision making is an important growth area within CEB that is unique in casting the widest net for cross-disciplinary cooperation and collaboration as well as providing a technology transfer opportunity to state and federal water and environmental agencies within California and beyond.
Direct biogeochemical analysis allows us to do structure and function studies of intrinsic microbial communities that take advantage of developments in the post-genomic era. These techniques have many applications to characterization, monitoring and control of biotreatment, bioremediation and natural attenuation processes. It also allows us to determine the microbial diversity in extreme environments that hold the genetic resources for novel traits and products of high economical value. The direct and real-time techniques that CEB is using also allow us to study biogeochemical mechanisms that have nanotechnology applications. Applications of synchrotron based spectromicroscopy enable us to understand the mechanisms of molecular and cellular processes for a wide variety of studies in environmental assessment including functional mechanisms in the environment and risk at the molecular level. At present, the Centers capabilities, expertise, and skills in the area of biogeochemical analysis include sample collection, microbial physiology, and molecular-level characterization. Our pioneering biochemical and biogeochemical imaging program using synchrotron-based x-ray and FT-IR spectromicroscopy techniques in conjunction with other molecular techniques has advanced in recent years to a cutting edge research capability. Chemical analysis and stable isotope investigation capabilities are available in the Environmental Measurement Laboratory (EML) and the Center for Isotope Geochemistry (CIG), respectively, that are closely associated with CEB.
Sample Collection. CEB researchers have considerable expertise in collecting environmental samples for biogeochemical analysis. They have collected pristine and contaminated field samples throughout North America, Europe and Asia. These samples have included gas, soil, sediment, and water from Caribbean coral reefs to sediments more than a mile deep in Siberian Lake Baikal. Indeed, CEB is extremely versatile in the types of field measurement it can take and the type of samples it is able to collect aseptically and preserve for direct measurements in the field or laboratory. Gaseous compounds or fluorescent polystyrene latex microspheres tracers are part of the sample collection protocols. For example, aseptically collected samples for biogeochemical analysis are split immediately: a subsample for isolation is kept at 4°C, while the rest of the sample is frozen and transported on ice, dry ice or in the vapor phase of liquid nitrogen for molecular-level characterization. Samples are kept at low or ultralow temperatures for long-term storage and a large variety of field controls, trip controls, internal standards, and experimental controls are used to produce defensible samples and analyses.Microbial Physiology. Molecular-level community characterization and an extensive isolation program go hand-in-hand. Our main objective with the isolation of microorganisms is to increase the number of cultured environmental strains and build a database with their physiological data and molecular-level traits to provide ground truth for our direct molecular measurements. A wide variety of isolation methods and low nutrient content media are employed. Incubation conditions (temperature, light, pH, Eh, etc.) simulate the eco-physiological conditions in the environment. Isolated environmental microorganisms, enriched cultures, genomic DNA samples, and clones are preserved and maintained in an ultralow temperature freezer at -86°C or in the vapor phase of liquid nitrogen.
Molecular-level Analyses. Since the majority of microorganisms that occur in the environment cannot be currently grown in the laboratory, alternative molecular-level techniques have been developed and applied widely in studying the structure and function of microbial communities in a given ecosystem. CEB has also developed a number of emerging physical/chemical techniques to directly assay biogeochemical changes in sediment and water at the molecular level. By combining these techniques, CEB intends to develop a better of understanding of dynamic and functional biogeochemical processes in the environment. CEB has the following expertise in this area:
DNA Sequencing and T-RFLP Analysis. The Center has a Model 377 ABI Prism automated DNA sequencer (Perkin Elmer). We perform dye terminator DNA sequencing using ABIs energy-transfer "Big Dyes" and cycle sequencing. There are four thermal cyclers available for running the cycle sequencing reaction. Terminal restriction fragment length polymorphism (T-RFLP) is a fairly recent fingerprinting technique. We use this pattern recognition technique to describe the microbial community structure based on sequence differences in the small subunit ribosomal RNA (rRNA) coding genes of the community members. Fatty Acid Methyl Ester (FAME) Analysis. The Center has a MIDI system, a dual tower GC/MS dedicated to fatty acid methyl ester analysis. Sample preparation and fatty acid methyl ester separation protocols follow the manufacturers recommendations. The available databases allow the comparative identification of pure cultures. Communities are also compared based on their GC chromatograms. The MIDI software enables us to build our own database for environmental cultures and communities.Signature Lipid Biomarker Analysis. Signature lipid biomarker (SLB) analysis as a complementary technique to nucleic acid analysis was developed to provide phenotypic information. SLB analysis provides insight into the viable biomass, total biomass, community structure, and nutritional/physiological status of the communities. The solvent extraction step was recently speeded up radically by the purchase and installation of a Dionex ASE 200 accelerated solvent extractor. Methylated fatty acids are separated on the MIDI system using the MIDI software Sherlock or HPs ChemStation. The former allows for the automatic identification of fatty acids up to C-20.Synchrotron-Based X-Ray and FTIR and X-Ray Spectromicroscopy Techniques. The high brightness of Synchrotron radiation-based (SR) Fourier-transform infrared (FTIR) spectromicroscopy at ALS allows our researchers to nondestructively map and track the progress of chemical reactions within a living biological system at a spatial resolution of 3-10 µm. This pioneering technique has been used successfully to probe and map the progress of attachment of microorganisms on mineral surfaces and the associated biochemical changes, the pathways of biogeochemical transformation of environmental contaminants on mineral surfaces, as well as the cellular responses to different doses of exogenous or endogenous stimuli. The nanoscale spatial resolution of x-ray microscope facilities (also at ALS) allow one to study the clustering and functions of biomolecules inside individual bacteria and cells.
Stable Isotopic Analysis. In collaboration with the Center for Isotope Geochemistry, CEB can examine a number of elements for their stable isotopic composition. This enables us to measure changes in environmental composition of contaminants, daughter products and a wide variety of electron donors and acceptors important in biogeochemical processes. Indeed, by combining these techniques with bio-molecular techniques like SLB we can even determine which functional part of a microbial community is using a particular substrate.
Environmental Risk Assessment. Direct analysis of real-time exposure and response of the same individual living human cells is the key to develop a better scientific basis for understanding exposures and risks to humans. Especially exposures from low dose radiation, known environmental pollutants, as well as newly synthesized chemicals. This can be achieved by the application of the synchrotron radiation-based (SR) Fourier transform infrared (FTIR) spectromicroscopy in conjunction with other cell molecular biology techniques. This is because SR FTIR spectromicroscopy is a novel and sensitive analytical tool for identifying and charting the time course of chemical changes in cellular nucleic acids and proteins of individual cells. At present, the Center's staffs are working with researchers from areas of biology, biomedicine, and physics to advance this approach to a cutting edge research capability. For example, we currently have found several spectral features that correlate extremely well with the expression of the CYP1A1 gene in response to polycyclic aromatic hydrocarbon and organochlorine exposures, but we do not yet know which part of the gene expression sequence is responsible for the specific vibrational modes being measured. By identifying if these spectral markers occur in the cell nucleus or within the cytoplasm, a better understanding and the development of a rapid, efficient diagnostic tool will result. We plan to achieve these goals by developing a sub-wavelength resolution near-field IR tool. This would allow one to map at 1-micron spatial resolution. Localization of the biological processes within the cellular compartments will therefore be achieved enabling a more complete understanding of the response mechanisms.
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