1. Microbial Ecology of the Centralia
Pennsylvania Mine Fire: The Study
of Thermophilic Actinomycetes
Kinley Hardy
Advisor: Dr. Tammy Tobin

2. History of Centralia, Pa
Trash fire which
started in 1962, still
burning today
Estimated to cover
400 acres on four
fronts
Ghost town today
Few residents
remain
Zip code
revoked in 2002

3. Why here?
 Fires have major
impacts on
ecosystems and
microbial communities
 Underground fire vs.
surface fire
 Duration of fire
 Unique chemical
composition and
temperature of soil

4.  New species and/or genera of thermophilic
actinomycetes will be discovered from
Centralia soil
 The probability of finding a novel species will
be greatest as the soil temperature rises
 Based upon previous research, the
thermophilic bacteria may have antimicrobial
or thermostable properties

5. What are Actinomycetes
 Gram positive, aerobic
bacteria
 Three genera:
Actinomyces, Nocardia and
Streptomyces
 Characteristic branching,
filamentous growth
 Spore forming

6.  Commonly found in soil
 Nitrogen fixation, decomposers, antimicrobial
production
 Medicine
 Commercially produced antibiotics
 Enzyme inhibitors
 Immunomodulators
 Agriculture
 Plant biotechnology
General Importance of
Actinomycetes

7. Methods
O Soil was collected from
three boreholes: 32.22˚C,
48.88˚C, and 65.55˚C
O Initially streaked on
glycerol yeast agar plates
or nutrient agar plates
O Isolates identified and
cultured
O DNA analysis
O PCR and Gel
electrophoresis
O BLAST
O Metagenomic Analysis

8. What is Metagenomics?
• The study of
metagenomes,
genetic material
recovered directly
from environmental
samples
• The genomic study of
uncultured microbes
• Pyrosequencing
• MacQIIME
(Quantitative Insights
into Microbial Ecology)

9. Table 1: Identified Isolates From Fall ‘13 and
Spring ‘14
Sample ID
Temperature
Collected (˚C)
Species with
closest 16S rRNA
match
% identity
Unknown 1 65.5 Brevibacillus sp. 99%
Unknown 2 65.5 Geobacillus sp. 96%
Unknown 3
32.22, 48.88 &
65.55
Bacillus fumariolo 99%
Unknown 5 32.22
Streptomyces
mexicanus 99%
Unknown 6 32.22 & 48.88
Bacillus gelatini
99 %
Unknown
A,C,D,E, &F
%

10. Figure 1: Illustrates the relative
abundance of each of the microbial rRNA
sequences extracted from the soil at
borehole 2 (60˚C). Larger bands directly
correlate to larger percentages of each
species present in the soil. The brown
band towards the top of the figure
represents Geobacillus sp. with an
abundance of 3.5%.
Figure 2: Section of phylogenetic tree from
MacQIIME analysis illustrating the relatedness of
Geobacillus to all other microbes extracted from
Centralia soil from all three temperature
boreholes. The branches are color coded to
indicate temperature extracted from. The
branch highlighted is Geobacillus.

11. Evidence of a Novel Species?
Figure 3: Gram stain of unknown 2
showingGram positive rods.
Figure 4: Endospore stain of
unknown two showing spore
formation

12. +
Additional Evidence
 Known to produce thermostable
enzymes and antimicrobials
 Grow optimally at temperatures
above 55˚C
 Can be cultured in TSA (tryptic
soy agar/broth)
 Spore-forming
 Generally aerobic, Gram
positive
 Produces a zone of inhibition
toward soil commensal
microbes
 Grows at 55˚C and isolated
from soil at 60˚C and above
 Can be cultured in tryptic soy
soil extract broth
 Spore-forming
 Aerobic, Gram positive
Characteristics of Geobacillus
species
Characteristics of Unknown 2

13. Future Research
Antimicrobial testing on identified isolates
Focus on unknown 2
Characteristics of unknown 2
morphology
Metabolism
Identification

14. Acknowledgements
 Dr. Tammy Tobin
 Laura Kraker
 Alex Rebuck
 Alysha Melynk
 COB students
 Peter Lynch
 Joneisha Moore
 Rebecca Kemmerer
 Lauren Kraemer

15. References
• DiSalvo, A. “Mycology - Chapter Two Actinomycetes.” Microbiology and
Immunology Online University of South Carolina School of Medicine. 27 October,
2010. Web. 2 December,
2013. http://pathmicro.med.sc.edu/mycology/mycology-2.htm
• Janzen, Chris and Tammy Tobin-Janzen. Microbial communities is fire-affected soils,
2008. Soil Biology, 13: 299-316.
• Nazina, T. N., Tourova, T. P., Poltaraus, A. B., Novikova, E. V., Grigoryan, A. A.,
Ivanova, A. E., ... & Ivanov, M. V. (2001). Taxonomic study of aerobic thermophilic
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stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus
kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations
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Microbiology, 51(2), 433-446.
• Sung, M. H., Kim, H., Bae, J. W., Rhee, S. K., Jeon, C. O., Kim, K., ... & Baek, D. H.
(2002). Geobacillus toebii sp. nov., a novel thermophilic bacterium isolated from
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52(6), 2251-2255.
• Waksman, S. A., Schatz, A., & Reynolds, D. M. (1946). Production of antibiotic
substances by actinomycetes. Annals of the New York Academy of Sciences, 48(2),
73-86.
• Wooley, J. C., Godzik, A., & Friedberg, I. (2010). A primer on metagenomics. PLoS
computational biology, 6(2), e1000667.
• Zeigler, D. R. (2001). Bacillus genetic stock center catalog of strains, seventh edition
volume 3: The genus geobacillus. (Doctoral dissertation, The Ohio State University)
Retrieved from http://www.bgsc.org/_catalogs/Catpart3.pdf