Grantee Research Project Results
2002 Progress Report: Microbial Biofilms as Indicators of Estuarine Ecosystem Condition
EPA Grant Number: R829458C002Subproject: this is subproject number 002 , established and managed by the Center Director under grant R829458
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: EAGLES - Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico
Center Director: Brouwer, Marius
Title: Microbial Biofilms as Indicators of Estuarine Ecosystem Condition
Investigators: Lepo, Joe , Proctor, Lita , Snyder, Richard
Institution: University of West Florida , University of Southern Mississippi
EPA Project Officer: Packard, Benjamin H
Project Period: December 1, 2001 through November 30, 2005 (Extended to May 20, 2007)
Project Period Covered by this Report: December 1, 2001 through November 30, 2002
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text | Recipients Lists
Research Category: Water , Aquatic Ecosystems , Ecological Indicators/Assessment/Restoration
Objective:
The main objective of this research project is to determine whether the microbial diversity of key microbial functional guilds will be lower in nutrient-impacted estuarine systems and higher in pristine estuaries.
Progress Summary:
General Considerations. Biofilm sampler design has been refined and the necessity to sample at different scales for different parameters is being addressed. Standard Operating Procedures (SOPs) for assays have been developed and are being assessed for their utility and information content for routine biofilm monitoring. We continue to facilitate collaborative research efforts with the scientists at the Gulf Breeze U.S. Environmental Protection Agency (EPA) research laboratory.
The spatial variability that one might encounter among biofilms generated on our sampling plates has been addressed. Results suggest that there is little variation within a single sampler, but we are finding some variation between biofilms in separated samplers. The habitat fidelity hypothesis testing has clearly demonstrated differences between habitats. This spatial variability will be addressed in the coming year as part of the indicator information available from biofilm samples.
Phospholipid fatty acid (PLFA) extraction and analysis SOPs have been completed. Fluorescein diacetate hydrolysis as a measure of total heterotrophic activity within biofilms is being developed. Progress continues on developing activity SOPs (acetylene reduction, nitrification/ denitrification) by adapting existing methods from the published literature.
DNA Molecular Biology Approaches. Postdoctoral Research Associate Andreas Nocker now routinely applies his improved DNA extraction procedure, which has been favored on the basis of yield, to biofilm samples. The lysis protocol includes sonication, enzymatic cell wall digestion, detergents, and freeze-thaw cycles. The DNA is subsequently extracted with phenol before precipitation. The first preliminary terminal-restriction fragment length polymorphism (T-RFLP) patterns suggest that up to 25 individual peaks (corresponding to the same number of individual species) can be distinguished in the samples. All genomic DNA extracted from the 2002 sampling round will be analyzed in the same way. The comparison of peak patterns will enable us to study the spatial diversity.
The Proctor laboratory is focusing on the microorganisms key to nitrogen transformations in aquatic systems such as nitrogen fixers, denitrifiers, and nitrifiers. These microbial groups are characterized by amplifying their functional genes, or genes that code for specific metabolic processes, from environmental DNA. nirS and nirK serve as probes for denitrifiers, while nifH probes for nitrogen fixers and amoA for nitrifiers. Our working hypothesis is that microbial diversity of key microbial functional guilds will be lower in nutrient-impacted estuarine systems and higher in pristine estuaries. Microbial diversity will be evaluated by T-RFLPs, and by sequence analysis of microbial biofilm clone libraries. Abundances of specific microbial guilds also will be estimated by quantitative real-time polymerase chain reaction (QPCR), using cultured species of nitrogen fixers, denitrifiers, and nitrifiers as standards.
Dr. Proctor's laboratory received biofilm DNA extracts from the 2002 field experiments in Pensacola Bay for use in her DNA molecular work. Independently, Drs. Nocker and Proctor have run test PCRs on the genomic DNA templates to assess the quality of the DNA. Dr. Proctor's laboratory is evaluating the quality of kit-extracted biofilm DNA versus manually extracted biofilm DNA by QPCR and by T-RFLPs of the functional genes nirS, nirK, amoA, and nifH. The ultimate comparison will be based on differences in the relative proportion of different functional guilds of microorganisms, as determined by QPCR, of the different biofilm preparations and by relative differences in the diversity of functional guilds, as determined by T-RFLP analyses, of the different biofilm preparations.
Dr. Proctor's laboratory has been refining the methods for amplifying functional genes from the DNA extracts of biofilms collected from Pensacola Bay and currently is in the process of developing clone libraries of the functional genes from the same material. We are using the TA TOPO cloning kit, which has higher cloning efficiencies than other cloning kits and also accepts smaller inserts than other cloning vectors. The clone sequences also will assist us in identifying the dominant members of each functional guild, which is based on the relative peak height of each terminal restriction fragments (t-RFs) in the T-RFLP profiles. The clone sequences also will be used to identify universal members of each functional guild across all estuaries under study as well as members unique to each type of estuary or estuarine habitat. We also have tested the QPCR protocols to optimize the linear range of response of the instrument to obtain the most reproducible estimates of microbial abundances in the biofilm extracts. We also are developing a direct count method with the DNA stain, 4',6-diamidino-2-phenylindole (DAPI), to enumerate total bacteria in formalin-preserved biofilms collected from Pensacola Bay.
PCR conditions for detecting various bacterial species (including sulfate-reducing bacteria [SRB], purple phototrophic bacteria, green sulfur bacteria, heliobacteria, and eubacterial dechlorinators) have been optimized with the help of genomic DNA from pure cultures as positive controls. The presence of these target groups will be quantitatively addressed using QPCR technology at a later stage. Other target groups, which will soon be added to the list, are green nonsulfur bacteria, urea-utilizing bacteria, methanogens, and methanotrophs.
One project has been started to analyze bacterial diversity in biofilms using a sequencing approach. Biofilms from two sites are being compared in regard to spatial differences in biodiversity: biofilms from an oyster-reef setting and biofilms grown at a local nonoyster reef. Genomic biofilm DNA from both locations has been isolated and has served as a template to amplify 16S rRNA genes using universal eubacterial primers. The PCR products were directionally cloned to construct a clone library. Individual clones were screened for the presence of an insert and were preselected with the help of RFLP. Unique clones will soon be sequenced to provide species information. The sequences also will help to assign individual peaks from community T-RFLP patterns. The latter are intended to provide "snapshot pictures" of bacterial biofilm communities at a later stage.
Another project similar to the study of diversity dynamics at one selected location has been assigned to graduate student Joe Moss. Samples will be collected every month over a 1-year period to study temporal changes in diversity. This will enlarge our own laboratory-generated clone library and provide more sequence data, making T-RFLP peak analysis more precise. The primary focus of Tim Huggins’ thesis, on the other hand, will be to analyze T-RFLP data with the help of the RDP database (making use of their rapidly accumulating 16S rRNA sequence data). This in silico approach will complement our laboratory-generated diversity data. Both approaches can be used to study the effects of different parameters on biodiversity in the planned microcosm experiments and to evaluate the suitability of biofilms as an environmental indicator. Initial indications are that this approach will provide greater resolution for determining community structure than the lipids analysis, but also at the cost of more intensive work. The additional information gained, however, and the rapidly advancing technologies for molecular analyses, make this portion of the project exciting in terms of indicator information content.
At the 2002 All-Estuarine and Great Lakes Program (EaGLes) meeting in Annapolis, MD, collaboration was initiated with Trish Holden’s laboratory (Pacific Estuarine Ecosystem Indicator Research Consortium, University of California, Santa Barbara) by exchanging information about biofilm sampling techniques and T-RFLP analysis.
Biofilm Sampler Deployments (Experiments). We have finished PFLA and C:N analyses of the biofilms from the sea grass experiment. There were no apparent differences in PFLA profiles of biofilms generated on floating samplers (1.25-m depth). Benthic samplers along a transect from near shore (1-m depth) midbed coincided with the floating sampler (1.25-m depth) and near the offshore edge of the bed (1.5 m). These initial results suggest that microbial communities developed over the spatial extent of this healthy system were consistent with one another.
To further assess habitat fidelity of biofilms, biofilm samplers were deployed in intertidal habitats, saltmarsh habitats, open sand flats, seagrass beds, and oyster reefs versus mud and sand bottom environments. Biofilms (physical and chemical parameters) and the resulting microbial communities are being evaluated from samplers in all of these environments. Ultimately, the habitat-specific biofilm data will be used to test the hypothesis of habitat fidelity in the microbial community structures. Laura Pennington, one of our graduate students, is developing a master’s thesis around this work, which will be completed in the coming year. Initial review of the emerging dataset suggests that at the level of resolution provided by phospholipids analysis, and in some cases, even at the crude level of biomass accumulation, habitat fidelity exists for the microbial communities within these developed biofilms on artificial substrates.
One deployment/incubation period was set to coincide with collections by our collaborators at the University of Southern Mississippi (Rakocinski, Brouwer) around Garcon Point in the Pensacola Bay system.
Microcosm Experiments. These experiments are designed to elucidate threshold responses of developing biofilms under two different nutrient exposure regimes (continuous and pulsed). Biofilm sampler plates would be deployed in flow-through microcosms with natural seawater with no added nutrients, a steady-state elevated level of N and P, and intermittent pulses of elevated N and P. The purpose is to: (1) ensure that we can measure responses in biofilms developed under controlled conditions; (2) allow for the adjustment of physical parameters (plate size, number, etc.) and incubation times; and (3) establish thresholds (biomass) for some of the assays (particularly activities such as nitrogen fixation, sulfate reduction, and heterotrophic activity). The flow-through rate is tentatively set for a 50 percent turnover time of 24 hours or approximately 95 percent turnover within 4 days. Project Investigators Snyder and Lepo, in consultation with the EPA Gulf Ecology Division (GED) have set up a wet table outside the GED Wet Laboratory to hold three aquaria (approximately 150 L; one for each nutrients regime); they have plumbed the aquaria to receive unfiltered, salinity-controlled seawater from the Santa Rosa Sound.
Future Activities:
We will continue to determine whether microbial diversity of key microbial functional guilds will be lower in nutrient-impacted systems and higher in pristine estuaries.
EPA GED collaborators, under the cooperative agreement for microbial biofilms, include Rick Greene, Bill Walker, Rich Devereux, Skeet Lores, Michael Lewis, and Peter Chapman. Rick Greene is the primary liaison for the project, and he will provide expertise on nutrient impacts and will coordinate access to gas chromatography, high-performance liquid chromatography and other instrumentation. Bill Walker will coordinate Wet Laboratory space and facilities for pilot studies. Rich Devereux will facilitate the development of DNA/RNA molecular technologies (e.g., PCR, T-RFLP) to assess broad taxonomic groups (16S) and for biogeochemical functional groups (e.g., nitrogen-fixing bacteria, nitrate reducers, sulfate reducing bacteria (SRBs)). Skeet Lores will provide expertise on the impact of nutrient loading, low dissolved oxygen, and general ecosystem stress response and to coordinate sampling time and sites to mutual benefit. Michael Lewis will provide expertise on biofilms development gained from his periphyton assay work and will provide expertise on site contamination with metals and toxic compounds, and Peter Chapman will assist in the design of microcosm experiments and will provide expertise on the use of mass isotopes for C and N source.
Journal Articles:
No journal articles submitted with this report: View all 27 publications for this subprojectSupplemental Keywords:
population, community, ecosystem, watersheds, estuary, Gulf of Mexico, nutrients, hypoxia, innovative technology, ecoindicators, biomarkers, water quality, remote sensing, global information system, GIS, integrated assessment, risk assessment, fisheries, conservation, restoration., RFA, Scientific Discipline, ECOSYSTEMS, Geographic Area, Water, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, estuarine research, Ecosystem/Assessment/Indicators, Ecosystem Protection, Aquatic Ecosystem, Aquatic Ecosystems, Ecological Effects - Environmental Exposure & Risk, Environmental Monitoring, Ecological Monitoring, Ecology and Ecosystems, Biology, Gulf of Mexico, Ecological Indicators, monitoring, ecoindicator, ecological exposure, remote sensing, estuaries, estuarine integrity, Mobile Bay, microbial biofilms, Galveston Bay, Apalachicola Bay, estuarine ecoindicator, environmental indicators, environmental stress, estuarine waters, restoration, water qualityProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R829458 EAGLES - Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829458C001 Remote Sensing of Water Quality
R829458C002 Microbial Biofilms as Indicators of Estuarine Ecosystem Condition
R829458C003 Individual Level Indicators: Molecular Indicators of Dissolved Oxygen Stress in Crustaceans
R829458C004 Data Management and Analysis
R829458C005 Individual Level Indicators: Reproductive Function in Estuarine Fishes
R829458C006 Collaborative Efforts Between CEER-GOM and U.S. Environmental Protection Agency (EPA)-Gulf Ecology Division (GED)
R829458C007 GIS and Terrestrial Remote Sensing
R829458C008 Macrobenthic Process Indicators of Estuarine Condition for the Northern Gulf of Mexico
R829458C009 Modeling and Integration
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
3 journal articles for this subproject
Main Center: R829458
175 publications for this center
52 journal articles for this center