Bacterial BioBlitz in Twelve All Taxa Biodiversity Inventory Plots

Scientific Findings

                There are essentially six lines of work ongoing in my research lab, with the four students mentioned above having been aided by students in three of my courses (General Ecology, Methods of General Microbiology, and Microbial Ecology) and work-study students.  Below are these ATBI-related projects and the status of each.

1. Archaea within GSMNP: psychrophiles and the culture challenge

                With the outstanding work of Gina Parise, we have been able to take what started out as a first look for Archaea in the Park and discover a unique group of organisms adapted to life at cold temperature.  We have sequenced DNA from seven unique organisms and found three of them to be aligned within the archaeal kingdom Euryarchaeota and four in the kingdom Crenarchaeota, the latter being representatives of the “non-thermophilic” branch of that kingdom.  The non-thermophilic Crenarchaeota have thus far eluded culturing by anyone and we are on the path to growing these organisms in the lab.  An experiment has been set up using humic acids in a growth medium at 4°C and we have observed prokaryotic cells increase in number in these cultures.  We have yet to identify the growing cells as our reliance upon PCR makes detection difficult since humics interfere with the PCR reaction.  However, we have dosed samples with one anti-fungal compound (cycloheximide) and three anti-bacterial agents (chloramphenicol, ampicillin, and streptomycin) as a means of eliminating organisms that are not archaeal.  A sample has been submitted to Microbial Insights in Knoxville to be analyzed for archaeal phospholipid fatty acids (PLFA), which will give us a definitive means of determining whether the cells we are growing are archaeal or bacterial (or both).  If we detect an archaeal signature, work will go forward on trying to isolate some of the species.     

                Sites sampled: Albright Grove ATBI plot (soil), Cataloochee ATBI plot (soil), Purchase Knob ATBI plot (soil), Beech Flats Prong/Minnie Ball Branch area (soil)

                4 Talks, 4 Posters, 7 Species identified, 1 Manuscript in progress

2. Bacterial biogeography: caves, streams, and forest soils

                Kristina Reid has led the charge on this project and along with 32 students in my Methods of General Microbiology course, has produced a great body of work.  Six bacterial kingdoms have been represented in our work and 52 total species identified.  We set out to determine in my General Ecology course of spring 2002 whether microbial communities within three forested ecosystems differed from each other based on forest history. Albright Grove (old growth forest), Purchase Knob (formerly logged forest), and Cataloochee ATBI (forest affected by Chestnut Blight) plots were sampled and additional samples from a cave, streams, and other forest soils were taken.  Kristina has shown there to be profound differences in the culturable organisms from each of the environments sampled, with 139 bacterial isolates being found in total.  Of these 139, 92 were different species and only one was found in all three ATBI plots and in the waters and sediments of Gregorys Cave.  Nearly 75% of all species were found in only one environment.  Kristina has earned a 2003-2004 Undergraduate Research Fellowship from the American Society for Microbiology to continue this work and a recent DLIA grant will help in sampling additional ATBI plot locations.       

                Sites sampled: Albright Grove ATBI plot (soil), Cataloochee ATBI plot (soil), Purchase Knob ATBI plot (soil), Gregorys Cave (sediment and water), Juney Whank Falls (soil and water), Kephart Prong (soil, stream sediment, and water)

                3 Talks, 5 Posters, 52 Species identified, 110 Species identifications in progress, 1 Manuscript in progress (a second one is possible)

3. Bat and bird microflora

                In a project in its infancy, I tried to detect microbial species in the feces of bats (Little Brown, Big Brown, and Eastern Pipistrelle) and birds (Eastern Towhee and Veery).  I was unsuccessful in my attempt to isolate DNA from all five species, but hope to try again with a different method.

                Sites sampled: Bats from Cades Cove during the 2002 Bat Blitz and birds from Purchase Knob as part of the Monitoring Avian Productivity and Success (MAPS) station work in 2002.

4. Extremophiles: Life at high temperatures

                Having discovered “cold-lovers” in the Park, Gina and Kristina looked at the other end of the temperature scale.  They designed an experiment to examine whether thermophiles existed in the Park.  Thermophiles are organisms that grow at temperatures ranging from 45 to 80°C  (113 to 176°F) and have an optimum growth temperature of 60°C.  Soil from three ATBI plots was examined from beneath five tree species and one gram of each soil sample inoculated into a basic microbiological growth medium (trypticase soy broth) and incubated at 65°C for a month. Results indicate that not only do thermophiles exist in the Park but they also appear to be quite common.  Thermophiles were detected from 16 of 21 samples taken in the Albright Grove, Purchase Knob, and Cataloochee ATBI plots.  In the nine soil samples taken from beneath Eastern Hemlocks (three from each ATBI plot), eight samples contained thermophiles.  At Albright Grove, one of three American Beech soil samples and two of three Rhododendron samples exhibited thermophilic growth.  Cataloochee samples showed growth at 65°C for two of three hemlock and Northern Red Oak samples each.  Lastly, all six samples from Purchase Knob (three from a birch and three from a hemlock) were positive for thermophily.  Results were gathered by visual observation of turbidity in media tubes and confirmation of cells was undertaken via microscopy, where the dominant cell shape was bacillus (rod-shaped).  This summer, a graduate student, Chris Le Moine, will confirm the identity of these thermophiles and determine whether they are Bacteria and/or Archaea. 

                Sites sampled: Albright Grove ATBI plot (soil), Cataloochee ATBI plot (soil), Purchase Knob ATBI plot (soil)

5. Microbial decomposition in Gregorys Cave: a possible link to forensics

                This work, conducted largely by Kim Lowery, set out to determine the bacterial dynamics of animal tissue decomposition in a cave ecosystem.  Kim has an interest in forensics and we wanted to tie her Senior Thesis into something ATBI-related so we settled on Gregorys Cave as a sample site (based on arthropod and vertebrate diversity work that we became familiar with showing three distinct zones in the cave).  The goal of the project was to eliminate eukaryotes (largely fungi and arthropods) from pieces of beef that were placed in Gregorys Cave and to monitor decomposition of the meat over time and between three locations in the cave.  After much trial and error, we were successful in preventing eukaryotic access to the meat samples (using “eukaryote guards” – stainless steel tea infusers surrounding skewered meat covered with cave soil that had been treated with cycloheximide and the whole infuser covered with two layers of nylon material).  Results show no difference in rates of decomposition or numbers of microorganisms on the meat samples, but a difference in communities of microorganisms between cave location and time.  These community patterns are currently being analyzed and species will be identified this summer and fall.  This work has caught the attention of the press department at the American Society for Microbiology and may prove to be useful in a newly developing field called “microbial forensics”.     

                Sites sampled: Gregorys Cave (organisms from soil spiked onto meat cubes incubated in the cave for up to three weeks).

                1 Talk, 3 Posters, 20 Species identifications in progress, 1 Manuscript in progress, 1 WCU Senior Thesis in progress (expected completion in Fall 2003)

6. Microbial response to stress: Beech Flats Prong

                In a project aimed at using microorganisms as environmental monitoring agents, Henry Angelopulos examined aqueous bacteria from Beech Flats Prong and Mille Ball Branch.  Beech Flats Prong has an ongoing input of acidic, metallic waste entering it from the Route 441 roadfill below Newfound Gap.  Henry sampled stream sites immediately above the roadfill, immediately below it, and one mile downstream, as well as in the proximal stream, Millie Ball Branch, which joins Beech Flats Prong at the downstream site.  Henry used many methods to describe the communities from the steams and found no difference in diversity for the four sites when using traditional diversity estimates of culturable bacterial patterns.  However, similarity indices using the same data sets showed remarkably different communities between the four sites in May and October of 2002.  Community-level techniques reflecting metabolic differences in the communities indicated that microorganisms from each of the four sites were different in their carbon source utilization patterns and ability to use ammonia and sulfur.  Organisms cultured from the sites will be identified this summer and fall.

                Sites sampled: Beech Flats Prong above and below the Route 441 roadfill and Minnie Ball Branch near the confluence with Beech Flats Prong (water)

                1 Talk, 2 Posters, 40 Species identifications in progress, 1 WCU Master’s Thesis completed (Spring 2003)