Florida Coastal Everglades Long Term Ecological Research
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Biogeochemical Cycling Working Group
(Phase II, 2007-2012)

Hypotheses and Proposed Work for FCE II (2006-2012)

General Question: How do water residence time and the magnitude of nutrient inputs, primarily from freshwater inflows, marine inputs, and groundwater, control local nutrient concentrations and cycling rates in the oligohaline ecotone?

Specific Research Question 1: What are the mechanisms by which P availability acts to regulate N cycling rates in marshes and mangroves of the southern Everglades?

Approach - Samples of soils, "floc", and periphyton will be collected during both wet and dry seasons from key ecotone sites and incubated in the laboratory. Nitrification potential will be measured using allylthiourea, which blocks nitrification (Ginestet et al. 1998). We will measure denitrification using acetylene block technique in samples treated with chloramphenicol, which prevents overestimation of denitrification rates (Bernot et al. 2003), and will compare this method to denitrification estimates obtained from membrane inlet mass spectrometry (Kana et al. 1998). N fixation potential will be measured using acetylene reduction and calibrated with 15N techniques (Seitzinger and Garber 1987). These N cycling processes will be correlated to the d15N content of soils, “floc”, and periphyton to determine how d15N values are regulated by processes such as fixation of atmospheric N and selective 15N uptake by microbes. Specifically, we will calibrate our N-fixation methods in Year 1, examine the effects of P additions on N-fixation (Year 2) and nitrification potential (Year 3), calibrate our denitrification methods (Year 4), examine the effects of P additions on denitrification (Year 5), and quantify P-addition effects on all 3 processes simultaneously (Year 6).

Specific Research Question 2: How is the soil bacterial community influenced by temporal changes in water source in the oligohaline ecotone, and how are these community shifts reflected in ecosystem processes, such as those of the N cycle?

Approach - We will use functional genomic analysis to elucidate bacterial community structure in soils and microbial mats at the TS/Ph and SRS oligohaline ecotone sites. DNA analysis may be applied at different levels of resolution: whole communities, bacterial isolates, and clones of specific genes. Low resolution and broad scale analysis of community DNA, like DNA-reassociation, allow the assessment of the total genetic diversity of bacterial communities (Torsvik et al. 1996). PCR combined with denaturing gradient gel electrophoresis (DGGE) analysis of rDNA operates at somewhat higher resolution, providing information about changes in the gross community structure (Muyzer et al. 1993). When DGGE analyses of 16S rDNA are combined with sequencing, assessment of the phylogenetic affiliation of the numerically dominant members of a community is obtained. Subsequent cloning of PCR products from 16S rDNA in whole community DNA provides significant new information about non-cultured bacteria. This approach also allows comparison of the structure of the previously cultivated fraction of a bacterial community with the total community. Finally, to discriminate at the bacterial isolate and clone levels, combined DNA fingerprinting and sequencing have been used (de Bruijn 1992, Massol-Deya et al. 1995, Stackebrandt and Rainey 1995). For our work, we will quantify organisms containing specific functional genes by real-time quantitative PCR (qPCR; i.e. for nitrogen fixation, nitrification, denitrification, and carbon fixation), and the relative expression of these select genes of ecological relevance from soils and microbial mats using quantitative real-time reverse transcription PCR (qRT-PCR). We will relate microbial community characterization to the process-based work of Question 4.1 by making these measurements in the same cores used for that work.

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National Science Foundation logo This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DEB-1237517, #DBI-0620409, and #DEB-9910514. Any opinions, findings, conclusions, or recommendations expressed in the material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
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