Microbial Community Assays

EPA Grant Number: R825433C045
Subproject: this is subproject number 045 , established and managed by the Center Director under grant R825433
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: EERC - Center for Ecological Health Research (Cal Davis)
Center Director: Rolston, Dennis E.
Title: Microbial Community Assays
Investigators: Scow, Kate
Institution: University of California - Davis
EPA Project Officer: Levinson, Barbara
Project Period: October 1, 1996 through September 30, 2000
RFA: Exploratory Environmental Research Centers (1992) RFA Text |  Recipients Lists
Research Category: Center for Ecological Health Research , Targeted Research

Objective:

This project seeks to develop methods for community measures of microbial structure and function and adapt methods for use in complex environmental media such as soil, groundwater, and sediments.

Approach:

Microbes are primary agents of decomposition and nutrient and energy flow in all ecosystems. Microbial food web interactions and population dynamics are potentially very complicated, with as many as 1000 species present in a gram of soil or sediment. Changes in microbial communities in response to stresses are poorly understood, primarily because they are difficult to measure. Previously existing methodology in microbial ecology has been dependent on culturing (most environmental species cannot be cultured), or lacked the sensitivity to detect changes in response to known biological stressors such as pesticides. New methods that allow detection of changes in microbial community structure and function will provide novel information about the potential of ecosystems to withstand stresses and decompose toxins. These methods are applicable to any ecosystem, both aquatic and terrestrial.

Phospholipid Fatty Acid (PLFA) Analysis. Phospholipid fatty acids (PLFAs) are integral components of cell membranes and are rapidly metabolized when a cell dies in soil; therefore, they provide an accurate measurement of living biomass. Phospholipids can be extracted directly from environmental samples to provide information about the microbial communities present. There are three major ways in which PLFA data can be used. First, the entire PLFA profile can be used as a "fingerprint" of the whole soil community. Signature lipids can also be used to indicate specific subgroups within the community. For example, signature fatty acids are associated with particular subsets of the microbial community, such as sulfate reducers, methane oxidizing bacteria, arbuscular mychorrizal fungi, and actinomycetes. Lastly, certain stresses, such as starvation, can induce changes in certain PLFA components such as the ratio of saturated to unsaturated fatty acids, ratio of trans to cis-monoenoic unsaturated fatty acids, and the proportion of cyclopropyl fatty acids.

Investigators have analyzed sediment microbial communities at different locations in Clear Lake. Microorganisms in mercury-polluted sediments (Clear Lake, CA) generate highly toxic and bioaccumulating methylmercury (CH3Hg+). Environmental variables likely to influence microbial methylmercury production include microbial community composition, Hg availability, carbon availability, redox potential, and availability of electron acceptors such as sulfate. However, the relative importance of these factors in controlling methylmercury production in situ is unknown. The potential role of microbial community composition was subsequently investigated through the analysis of phospholipid fatty acids (PLFAs) extracted directly from sediment cores. Spatial and temporal variation in PLFA profiles suggested that community structure may be an important control on methylmercury production. Lake location was the most important predictor of sediment microbial community composition. Seasonal changes, depth interval (0-8 cm), and changes potentially attributable to temperature regulation of bacterial membranes were detectable but much less important influences on sediment PLFA composition. Mercury methylation potential and sediment organic carbon content were significantly related to sediment PLFA composition, whereas pore water sulfate, total mercury concentrations and organic matter C/N rations were not. Mercury methylation potential per unit of microbial biomass was highest at lake locations which had the highest abundance of biomarkers characteristic of sulfate-reducing bacteria in the genus Desulfobacter, suggesting that Desulfobacter or sulfate-reducing bacteria with similar PLFA composition, are important mercury methylators in the Clear Lake sediments.

Development of nucleic acid methods. Two nucleic acid-based methods are being explored as means for characterizing microbial communities in soil: Denaturing Gel Gradient Electrophoresis (DGGE) analysis, and Intergenic Transcribed Spacer (ITS) analysis. DGGE involves the amplification of the 16S ribosomal gene fragment from the total microbial sediment community and the subsequent separation of these gene fragments on a polyacrylamide gel, resulting in a DNA fingerprint of the microbial community. The ITS analysis involves the amplification of the sequence between the small and large subunit ribosomal sequences. Microbial strains have unique ITS sequences that can range in size from 200 to 1400 base pairs. ITS analysis can distinguish closely related strains from each other because the spacer region is not an evolutionary conserved sequence. DGGE, on the other hand, can only separate distantly related organisms from each other. DGGE is currently being performed on mercury-contaminated sediment and floc samples using the conserved primers 9884F and 9885R.

Linking microbial community composition to metabolic functions. Though soil microbial communities are extraordinarily diverse, it is unknown what portion of the community is actively engaged in a specific process. Analysis of PLFAs can be used to quantitatively describe a community on the basis of its lipid composition. Analyzing 13C-labeled PLFAs resulting from incorporation of stable isotope-labeled C substrates into soil provides a way to define the groups of organisms utilizing those substrates. PLFA analysis of a soil microbial community was coupled with 13C isotope tracer analysis to measure the microbial community's response to the addition of glucose, utilized by a many microorganisms, and toluene, used by a relatively small subset of microorganisms. We hypothesized that 13C from glucose would become incorporated into a large number of the PLFAs extracted from the soil community, whereas only a small set of PLFAs would become labeled from the 13C-toluene.

Yolo silt loam was incubated with 35 µg [13C] toluene ml-1 soil solution. After 119 hrs incubation with toluene, 96% of the incorporated 13C was detected in only 16 of the total 59 PLFA (27%) extracted from the soil. Of the total 13C-enriched PLFA, 85% were identical to the PLFAs contained in a toluene-metabolizing bacterium (Rhodococcus sp.) isolated from the same soil. In contrast, the majority of the soil PLFAs (91%) became labeled when the same soil was incubated with [13C] glucose. Investigator's study showed that coupling 13C-tracer analysis with PLFA analysis is an effective tool for distinguishing a specific microbial population involved in metabolism of a labeled substrate in complex environments such as soil.

ITS analysis was used to determine the effect on the Yolo soil community of exposure to different concentrations of toluene. Among the DNA bands extracted from the soil community, one band migrated to the same position as DNA extracted directly from the Rhodococcus sp. described above. When the DNA in the band was excised, cloned, and sequenced, it was confirmed to be identical to the sequence in the ITS region of the Rhodococcus sp.

Expected Results:

Investigators are working to improve understanding of microbial community structure and function in three ways. The first major challenge is to improve the quantification of DNA bands on electrophoresis gels. Given that PCR amplification is involved with all of the methods used, there is potential for artifacts or distortion of band intensity in community DNA fingerprints. Second, they hope to expand the DGGE analyses to include the use of specific bacterial primers which identify particular bacterial groups within a community, such as sulfate-reducers or ammonia-oxidizers. A final task is to link PLFA biomarkers to DNA sequences to provide a better understanding of community composition.

Supplemental Keywords:

Watershed, bioindicator, biomarker, ecosystem stress, Clear Lake, food web, mercury methylation, microbial ecology, microbial assays., RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Geographic Area, Water, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Water & Watershed, Ecosystem/Assessment/Indicators, exploratory research environmental biology, Environmental Chemistry, State, Aquatic Ecosystem, Ecological Effects - Environmental Exposure & Risk, Monitoring/Modeling, Biochemistry, Environmental Monitoring, Ecological Monitoring, Ecology and Ecosystems, Ecological Indicators, Watersheds, nutrient dynamics, watershed development, marine food web, wetlands, immunoassay, Clear Lake , ecosystem monitoring, watershed management, fish habitat, biomarkers, anthropogenic effects, agricultural watershed, microbial assays, runoff, aquatic habitat, Clear Lake, watershed land use, watershed modeling, microbial ecology, ecological assessment, hydrology, integrated watershed model, aquatic ecosystems, environmental stress, lake ecosysyems, bioassay, water quality, watershed sustainablility, ecological risk, ecosystem health, ecosystem stress, California (CA), ecology assessment models, environmental stress indicators, water management options, wildlife habitat, ecosystem response, land use, watershed restoration

Progress and Final Reports:

1999 Progress Report
Final Report


Main Center Abstract and Reports:

R825433    EERC - Center for Ecological Health Research (Cal Davis)

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R825433C001 Potential for Long-Term Degradation of Wetland Water Quality Due to Natural Discharge of Polluted Groundwater
R825433C002 Sacramento River Watershed
R825433C003 Endocrine Disruption in Fish and Birds
R825433C004 Biomarkers of Exposure and Deleterious Effect: A Laboratory and Field Investigation
R825433C005 Fish Developmental Toxicity/Recruitment
R825433C006 Resolving Multiple Stressors by Biochemical Indicator Patterns and their Linkages to Adverse Effects on Benthic Invertebrate Patterns
R825433C007 Environmental Chemistry of Bioavailability in Sediments and Water Column
R825433C008 Reproduction of Birds and mammals in a terrestrial-aquatic interface
R825433C009 Modeling Ecosystems Under Combined Stress
R825433C010 Mercury Uptake by Fish
R825433C011 Clear Lake Watershed
R825433C012 The Role of Fishes as Transporters of Mercury
R825433C013 Wetlands Restoration
R825433C014 Wildlife Bioaccumulation and Effects
R825433C015 Microbiology of Mercury Methylation in Sediments
R825433C016 Hg and Fe Biogeochemistry
R825433C017 Water Motions and Material Transport
R825433C018 Economic Impacts of Multiple Stresses
R825433C019 The History of Anthropogenic Effects
R825433C020 Wetland Restoration
R825433C021 Sierra Nevada Watershed Project
R825433C022 Regional Transport of Air Pollutants and Exposure of Sierra Nevada Forests to Ozone
R825433C023 Biomarkers of Ozone Damage to Sierra Nevada Vegetation
R825433C024 Effects of Air Pollution on Water Quality: Emission of MTBE and Other Pollutants From Motorized Watercraft
R825433C025 Regional Movement of Toxics
R825433C026 Effect of Photochemical Reactions in Fog Drops and Aerosol Particles on the Fate of Atmospheric Chemicals in the Central Valley
R825433C027 Source Load Modeling for Sediment in Mountainous Watersheds
R825433C028 Stress of Increased Sediment Loading on Lake and Stream Function
R825433C029 Watershed Response to Natural and Anthropogenic Stress: Lake Tahoe Nutrient Budget
R825433C030 Mercury Distribution and Cycling in Sierra Nevada Waterbodies
R825433C031 Pre-contact Forest Structure
R825433C032 Identification and distribution of pest complexes in relation to late seral/old growth forest structure in the Lake Tahoe watershed
R825433C033 Subalpine Marsh Plant Communities as Early Indicators of Ecosystem Stress
R825433C034 Regional Hydrogeology and Contaminant Transport in a Sierra Nevada Ecosystem
R825433C035 Border Rivers Watershed
R825433C036 Toxicity Studies
R825433C037 Watershed Assessment
R825433C038 Microbiological Processes in Sediments
R825433C039 Analytical and Biomarkers Core
R825433C040 Organic Analysis
R825433C041 Inorganic Analysis
R825433C042 Immunoassay and Serum Markers
R825433C043 Sensitive Biomarkers to Detect Biochemical Changes Indicating Multiple Stresses Including Chemically Induced Stresses
R825433C044 Molecular, Cellular and Animal Biomarkers of Exposure and Effect
R825433C045 Microbial Community Assays
R825433C046 Cumulative and Integrative Biochemical Indicators
R825433C047 Mercury and Iron Biogeochemistry
R825433C048 Transport and Fate Core
R825433C049 Role of Hydrogeologic Processes in Alpine Ecosystem Health
R825433C050 Regional Hydrologic Modeling With Emphasis on Watershed-Scale Environmental Stresses
R825433C051 Development of Pollutant Fate and Transport Models for Use in Terrestrial Ecosystem Exposure Assessment
R825433C052 Pesticide Transport in Subsurface and Surface Water Systems
R825433C053 Currents in Clear Lake
R825433C054 Data Integration and Decision Support Core
R825433C055 Spatial Patterns and Biodiversity
R825433C056 Modeling Transport in Aquatic Systems
R825433C057 Spatial and Temporal Trends in Water Quality
R825433C058 Time Series Analysis and Modeling Ecological Risk
R825433C059 WWW/Outreach
R825433C060 Economic Effects of Multiple Stresses
R825433C061 Effects of Nutrients on Algal Growth
R825433C062 Nutrient Loading
R825433C063 Subalpine Wetlands as Early Indicators of Ecosystem Stress
R825433C064 Chlorinated Hydrocarbons
R825433C065 Sierra Ozone Studies
R825433C066 Assessment of Multiple Stresses on Soil Microbial Communities
R825433C067 Terrestrial - Agriculture
R825433C069 Molecular Epidemiology Core
R825433C070 Serum Markers of Environmental Stress
R825433C071 Development of Sensitive Biomarkers Based on Chemically Induced Changes in Expressions of Oncogenes
R825433C072 Molecular Monitoring of Microbial Populations
R825433C073 Aquatic - Rivers and Estuaries
R825433C074 Border Rivers - Toxicity Studies