2005 Progress Report: Microbial Biofilms as Indicators of Estuarine Ecosystem Condition

EPA Grant Number: R829458C002
Subproject: 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: Hiscock, Michael
Project Period: December 1, 2001 through November 30, 2005 (Extended to May 20, 2007)
Project Period Covered by this Report: December 1, 2004 through November 30, 2005
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text |  Recipients Lists
Research Category: Water , Ecosystems , Ecological Indicators/Assessment/Restoration

Objective:

Milestones for Year 4: (1) deployment and analysis of biofilms in Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico (CEER-GOM) targeted estuaries; (2) modeling and analysis of biofilm response to environmental variables; (3) reporting progress and discussion with other indicator groups; (4) evaluation of measured biofilm parameters for information content; and (5) technology transfer to environmental quality monitoring agencies.

During this reporting period, the following employees were working on microbial biofilms activities in laboratory and field experiments.

  • Postdoctoral Associates: Hugo Castillo, Song Hyon Lee
  • Technicians: Melissa Hagy, Alan Knowles
  • Graduate students: Joe Moss, Jeffrey Allison, Matthew Wagner
  • Undergraduate laboratory assistants: two

Progress Summary:

Research activity during this period included continuing analysis of samples collected from previous years, synthesis of data, completion of two master’s theses, microcosm work, a field experiment coordinated between biofilms and remote sensing projects, and continued manuscript writing and editing to publish results. Results from a previously completed master’s thesis have been published in the journal Applied and Environmental Microbiology. The two theses just completed are being reformatted and edited for publication.

Microcosms were run during the summers of 2005 and 2006 in the laboratory. Estuarine water, collected daily from the Pensacola Bay System, was used to replenish the supply carboys. Treatments were designed to simulate the effect of dissolved oxygen (DO) on biofilm growth and other endpoints under controlled conditions. Conditions were: (1) no added nutrients, vigorous aeration; (2) no added nutrients, no aeration; (3) nutrients, vigorous aeration; and (4) nutrients, no aeration.

We were able to maintain approximately 1 ppm more DO in the aerated microcosms for 3 to 5 days, after which biological oxygen demand (BOD) caused DO to drop in all microcosms. The following parameters from these experiments are currently being assessed at 4 or 5 days:

  • Pixel density of scans (average of three plates per treatment)
  • Dry weight of biomass post enzyme assay
  • Acetylene reduction for nitrogen fixation
  • Fluorescein diacetate hydrolysis for general esterase activity
  • Phosphatase (alkaline) activity
  • C and N
  • DNA for 16S and functional guild analysis

A coordinated sampling of Pensacola Bay with Luoheng Han’s remote sensing work (see report for R829458C001) was conducted, with the biofilms project analyzing biofilms and water quality parameters and providing logistical support for the field operations. A significant feature of this work will be a report on the use of hyperspectral scanning for non-destructive biofilm chlorophyll determination.

Interactions and Collaborations

CEER-GOM. A collaboration with Luoheng Han’s remote sensing work involved analysis of biofilms and water quality parameters, in addition to providing logistical support for stations along pre-arranged flight lines.

A data synthesis meeting at University of Southern Mississippi was attended and issues related to the larger group integration and collaboration in C enter-wide publications were addressed. Datasets have been transferred to the consortium data manager.

State of Florida. Staff from the Florida Department of Environmental Protection (FDEP) attended a Master’s thesis defense on biofilms. Subsequent discussions resulted in University of West Florida-FDEP collaboration on a grant request for water quality work in Pensacola Bay that includes the use of biofilms as sensors.

Summary of Significant Research Progress During the Reporting Period

Research activity during this period was dominated by continuing analysis of samples collected from previous years, synthesis of data, completion of two master’s theses, and continued manuscript writing and editing to publish results.

Molecular Indicators Within Biofilms

We have previously reported and/or published the following observations on molecular indicators within biofilms. Manuscripts are being prepared and analyses to fill identified data gaps are being pursued.

  • Biofilm communities from a seagrass bed and a sandflat habitat in Pensacola Bay, Florida. were compared using 16S rRNA genes and functional genes characteristic for sulfate reducing (dsr) and nitrogen cycling guilds (nifH, nirS, nirK, amoA) of prokaryotes. Sequencing of clone libraries representing functional guilds allowed assignment of individual terminal fragments to taxonomic unit s and revealed phylogenetic interrelationships with an aim to demonstrating habitat specificity of biofilms.
  • Terminal restriction fragment length polymorphism (TRFLP) analysis and sequencing of clone libraries from biofilm communities collected at sewage outfall showed prominent signature peaks (both 16S and dsr) that differed from those generated from a relatively pristine site with a strong effect of low DO.
  • Biofilms responded to hypoxic conditions in the field as indicated by TRFLP analysis elucidated a 91-bp TRF consistent with the δ-Proteobacteria, which includes the majority of sulfate reducing bacteria .
  • Without exception, biofilms exposed to hypoxic conditions show substantially more diverse dsr fingerprints compared to biofilms grown under aerobic conditions, a relationship apparent with all three restriction enzymes used.
  • We are currently gathering supporting data to develop publications on all of these observations.

Field Sampling

Pensacola Bay. We coordinated sampling with Luoheng Han at the University of Alabama at Tuscaloosa on the 16 U.S. Environmental Protection Agency Gulf Ecology Division Pensacola Bay open water stations. This work was to compare biofilm response to planktonic chlorophyll determined by remote sensing. Sample analysis for this work is complete and data analysis is underway to culminate in a manuscript comparing remote sensing with biofilm sensors and on the use of hyperspectral scanning as a means of determining biofilm chlorophyll content in the field. We are also in the process of discussing with an instrument company the possibilities for automating sensing of biofilm growth as chlorophyll and mass to couple a biological sensor with electronic sensors.

Microcosm Experiments. The physical setup of our microcosms and operating parameters were described in our 2005 annual report. Molecular data concerning the diversity of microbial guilds responsible for ammonia oxidation, nitrogen fixation, nitrate reduction, and sulfate reduction have been gathered for all microcosm experiments and are currently being analyzed to determine probable cause and effect relationships between nutrient bioavailability and biofilm community structure and function.

We have completed additional microcosm experiments designed to simulate the effect of DO on biofilm growth and other endpoints under controlled conditions. We used water gathered daily from Bayou Texar and other regional bays and bayous with salinity varying between 10 and 20 ppt. Conditions were: (1) no added nutrients, vigorous aeration; (2) no added nutrients, no aeration; (3) nutrients (NH4, NO3, PO4), vigorous aeration; and (4) nutrients, no aeration.

We were able to maintain approximately 1 ppm more DO in the aerated microcosms for 3 to 5 days, after which BOD caused DO to drop in all microcosms. Figure 1 shows typical microcosm DO profiles over 4 days. Thus these experiments were run only as long as we could maintain some differential between the aerated and non-aerated conditions.

Typical DO for Microcosms

Figure 1. Typical DO for Microcosms

Parameters Assessed for Microcosm Biofilms at 4 or 5 days:

  • Pixel density of scans (avg. of three plates per treatment)
  • Dry weight of biomass post enzyme assay
  • Acetylene reduction for nitrogen fixation
  • Fluorescein diacetate hydrolysis for general esterase activity
  • Phosphatase (alkaline) activity
  • C and N
  • DNA for 16S and functional guild analysis

Some obvious differences are that biofilms harvested from aerated microcosms consistently showed higher specific activity of fluorescein diacetate hydrolysis than did biofilms from the corresponding non-aerated microcosm, as shown in Figure 2.

FDA Hydrolysis as Millimole Fluorescein min-1  mg-1 Dry Weight of Biofilm

Figure 2. FDA Hydrolysis as Millimole Fluorescein min-1 mg-1 Dry Weight of Biofilm

Data f rom t his s et of m icrocosm e xperiments a re c urrently b eing a nalyzed.

The thesis work of Graduate Student Joseph Anthony Moss was published in September 2006 in Applied and Environmental Microbiology (71:5679). Biofouling communities contribute significantly to aquatic ecosystem productivity and biogeochemical cycling. Our knowledge of the distribution, composition, and activities of these microbially dominated communities is limited compared to other components of estuarine ecosystems. This study investigated the temporal stability and change of the dominant phylogenetic groups of the domain Bacteria in estuarine biofilm communities. Glass slides were deployed monthly over 1 year for 7-day incubations during peak tidal periods in East Sabine Bay, Florida. Community profiling was achieved by using 16S rRNA genes and T RFLP of 16S rRNA genes in combination with ribotyping, cloning, and sequencing to evaluate diversity and to identify dominant microorganisms. Bacterial community profiles from biofilms grown near the ben thos showed distinct periods of constancy within winter and summer sampling periods. Similar periods of stability were also seen in T RFLP patterns from floating biofilms. Alternating dominance of phylogenetic groups between seasons appeared to be associated with seasonal changes in temperature, nutrient availability, and light. The community structure appeared to be stable during these periods despite changes in salinity and in DO.

Graduate Student Matt Wagner successfully defended his thesis, Spatial Patterns of Phyto-plankton and Periphyton Growth as Indicators of Estuarine Condition in Escambia Bay, Florida and completed his Master of Science on July 13, 2006. The standard periphyton technique was employed to examine algal response at fixed points as 7- day integrated samples along the salinity gradient and in depth profiles in Escambia Bay, Florida, and these patterns were compared to the distribution patterns of phytoplankton biomass. Periphyton response allows determination of nutrient loading sources as opposed to hydrodynamic forcing controlling the accumulation of phytoplankton biomass a t the estuarine chlorophyll maximum. Vertical profiles indicate that water chemistry, not light, has an overriding effect on microalgal production, and that the periphyton assay is sensitive to hypoxia. Manuscripts from this thesis are in preparation.

Graduate Student Jeffrey Allison successfully defended his thesis, Importance of Microphyto-benthos at the Benthic Boundary Layer in an Estuarine Sand Bottom Habitat and completed his Master of Science on July 13, 2006. Microphytobenthos inhabiting subtidal estuarine sediments are important contributors to marine food webs and biogeochemical transformations of nutrients. He investigated production and respiration rates, biomass estimations, and nutrient flux rates in Butcherpen Cove, Pensacola Bay, Florida , from February through August 2005. Extracellular enzyme activity was assayed on slurries of periphyton grown on artificial substrates. Production and respi ration were measured by oxygen changes in light and dark incubations in situ. Microphytoben thos production and biomass decreased following major storm events. Water column phytoplank ton and periphyton did not respond to rainfall and appeared to be controlled by temperature. Although phytoplankton production was reduced in 2005 relative to previous reports, values in creased through the sampling season and variability was low. Periphyton grown on artificial substrates did not correlate with benthic processes but appeared to be responding to water column conditions. Bioavailability of water column nutrients did not seem to be as important to benthic production as much as benthic fluxes. The data suggest nitrogen and phosphorous nutrient co-limitations existed for both phytoplankton and benthic microalgae in Butcherpen Cove. When dissolved PO4-3 increased in the water column, alkaline phosphatase activity was suppressed. Esterase activity of biofilms on artificial substrates reflected biofilm community respiration. N-fixation and denitrification rates were minimal. Manuscripts from this thesis are in preparation.


Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 27 publications 4 publications in selected types All 3 journal articles
Other center views: All 171 publications 54 publications in selected types All 48 journal articles
Type Citation Sub Project Document Sources
Journal Article Moss JA, Nocker A, Lepo JE, Snyder RA. Stability and change in estuarine biofilm bacterial community diversity. Applied and Environmental Microbiology 2006;72(9):5679-5688. R829458 (2005)
R829458C002 (2004)
R829458C002 (2005)
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  • Abstract: Applied and Environmental Microbiology-Abstract
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  • Other: Applied and Environmental Microbiology-Full Text PDF
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  • Supplemental Keywords:

    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 quality

    Relevant Websites:

    http://www.usm.edu/gcrl/ceer_gom/ Exit

    Progress and Final Reports:

    Original Abstract
  • 2002 Progress Report
  • 2003 Progress Report
  • 2004 Progress Report
  • 2006
  • Final

  • Main 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