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.
