2004 Bennin, Andre Parker

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2004 Bennin, Andre Parker Powered By Docstoc
					                               Bennin, Andre
Comparison of Microbial Community Biodiversity between Pristine
   and Disturbed Sediments in Timpanogos Cave, National
                          Monument.
            Faculty mentor: Keith A. Crandall, Microbiology and Molecular Biology

Cave microbiology has been of great interest to microbial ecologists in recent years
because it has enabled them to assess microbial diversity within an isolated system. An
excellent example of subterranean microbial life is found in Lechuguilla Cave Carlsbad
Caverns National Park, New Mexico,where the interactions of microbial communities in
cave deep subsurface environments have been studied. With respect to the
Timpanogos Cave system in American Fork Utah, our study relates to a system that is
highly visited (60% of the cave system) contrary to the Lechuguilla cave system where
public access is only allowed into 2% of the cave system and tours are kept to a
minimum of 10-20 groups per year. Approximately 80,000 people each year introduce
mud, hair, lint, and debris into the Timpanogos cave system. The accumulation of these
foreign materials is a threat to the natural cave environment. They can change the
appearance of cave formations, add alternate energy sources allowing non-native
species to invade the cave ecosystem, add impurities to the cave water, and can
change the cave’s chemistry. This accumulation of foreign materials threatens the
preservation and conservation of the Timpanogos Cave System as a National
Monument.

The goal of our research was to document microbial communities in pristine and
disturbed areas of the cave. The specific objectives of this project are to: 1) characterize
the microbial biodiversity in different areas of the cave; 2) determine if the microbial
communities are different in pristine and disturbed sites; and 3) identify unique cave
microbial fauna. Comparisons between pristine and disturbed sites were made to see if
there were differences in community composition and demonstrate if large numbers of
tourists have affected the cave microbial ecosystem.

We collected samples from 10 different sites in the cave system. These samples
ranged from water, fungal, fine clay sediments, wood, and mud. Molecular based
surveys of 16S ribosomal DNA and phylogenetic analyses were used to determine
community composition and relative abundances of bacteria and archaea in the cave.
We extracted DNA out of the samples then performed Polymerase Chain Reaction
(PCR) on the samples we were able to extract DNA from. The samples that showed
bright bands were cloned, and then sequenced after a series of screening test. We
completed a phylogenetic analysis of the samples to determine the taxonomic affiliation
of each clone. In addition, the ribosomal database project
(http://rdp.cme.msu.edu/index.jsp) was used to identify chimeric sequence constructs
from the obtained sample sequences. Identified chimeric sequences were removed
from our phylogenetic analyses.
We have sequenced a total of 113 bacteria clones and 39 archaea clones. Five major
groups were identified for the obtained bacterial analyses, which include:
Gammaproteobacteria, Acidobacteria, Planctomycetia, Sphingobacteria and
Flavobacteria (Fig. 1). 55% of the obtained samples from disturbed site TC9R were
related to pseudomonas and gammaproteobacteria and the remaining 45% were
related to agricultural soil flavobacteria, suggesting a potential impact of microbial
communities within this site. 85% of the samples obtained from site TC8R (disturbed)
were related to uncultured soil bacteria. The remaining 15% were related to Agricultural
soil flavobacteria, suggesting a slight impact of this microbial community from foreign
non-native organisms. Samples obtained from site TC7R (disturbed) were closely
related to proteobacteria.

All of the obtained archaea samples have come from TC3.2R and T10R, which are
pristine sites; we have also obtained clones from TC7R, a disturbed site. Most of the
samples showed relatedness to crenarchaeotes and soil crenarchaeotes (Fig. 2). The
largest clone archaea group consisted of 70% mesophilic soil crenarchaeotes and front
range soil crenarchaeotes. Metal-rich associated archaea represent the remaining 30%
of our sampling. Tourist impact will be discussed when more archaea samples are
obtained from disturbed sites.

Additional samples were obtained from a new disturbed site on subsequent visits to the
cave. Four samples were obtained from this site and were related to environmental
proteobacteria Yersinia species.

Bacterial communities have shown to be more diverse than archaea communities in this
system. Results have also shown that community composition between sites is
different. We obtained archaea sequences from two pristine sites and they have shown
similarity in groups; however, we have not obtained archaea sequences for disturbed
sites, which prevent us from efficiently comparing the biodiversity between sites for
archaea samples. We are still in the process of sequencing more archaea samples in
order to make a better comparison of the archaea biodiversity in the cave system.
                             Timpanogos Cave Bacteria diversity



                                                                     Gammaproteobacteria
                                                                     Planctomycetia
                       4%                                            Acidobacteri a
                            4%    5%    1%
                   1%                                          33%   Deltaproteobacteria
                 11%
                                                                     Flavobacteria
                                                                     Shingobacteria
                                                                     Nitrospirales
                  11%
                            5%                           15%         Verrumicrobia
                                     10%
                                                                     Actinobacteridae
                                                                     Bacteroidetes
                                                                     Deinococci
                 Figure 1: Pie chart of bacterial diversity.
                 The tree could not be pictured due to
                 limited space.
          U. archaeon clone (AY354124)
         U. archaeon cl (AY354124)
            U. archaeon clone (AY354115)
    U. archeon cl(AY182126)
                                                               Haloarcula ajinwuensis(AY208973)
                                                           U.haloarchaeon(AF5069421)
                                                    U. ar clone(AY161250)
                                           U. ar Soyang(AF056368)
                                             U.ar gene clone (AB109885)
                                                              U. crenarchaeote cl(AF052949)
                                                              U. crenarchaeote clone(AY320199)
                                                          U. arch (UAU62819)
                                                            U. arch. clone (AY187899)
                                                           U. F.R soil crenarch (AY016505)
                                                          TC3 2R.52
                                                            U. crenarch(AJ496176)
                                                             U. F.R soil crenarch(AY016478)
                                                           U. archeon(U62811)
                                                           TC3.2R.69
                                                           U. crenarch (AF227643)
                                                           U. arch. (UAU62811)
                                                           TC3 2R.55
                                                           TC3.2R.70
                                                            TC3 2R.54
                                                           TC3 2R.57
                                                            U. archaeon clone(AF418935)
                                                             TC3 2R.56
                                                             U. crenarchaeote (AY487104)
                                                            U. crenarch. (AY487104)
                                                            U. ar clone(AY464752
                                                            U. archaeon clone(AY464752)
10 changes
                Figure 2: This is the archaeal tree with our samples pictured in red.