Microbial Ecology of the Centralia Pennsylvania Mine Fire: The Study
of Thermophilic Actinomycetes
Kinley HardyAdvisor: Dr. Tammy Tobin
History of Centralia, PaTrash fire which started in 1962, still burning today Estimated to cover 400 acres on four frontsGhost town today
Few residents remainZip code revoked in 2002
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
Hypotheses
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
What are ActinomycetesGram positive, aerobic
bacteriaThree genera:
Actinomyces, Nocardia and Streptomyces
Characteristic branching, filamentous growth
Spore forming
Commonly found in soil Nitrogen fixation, decomposers, antimicrobial
production Medicine
Commercially produced antibiotics Enzyme inhibitors Immunomodulators
Agriculture Plant biotechnology
General Importance of Actinomycetes
MethodsO 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 analysisO PCR and Gel
electrophoresisO BLASTO Metagenomic Analysis
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)
Table 1: Identified Isolates From Fall ‘13 and Spring ‘14
Sample IDTemperature Collected (˚C)
Species with closest 16S rRNA match % identity
Unknown 1 65.5Brevibacillus
sp. 99%
Unknown 2 65.5 Geobacillus sp. 96%
Unknown 332.22, 48.88 &
65.55Bacillus
fumariolo 99%
Unknown 5 32.22Streptomyces
mexicanus 99%
Unknown 6 32.22 & 48.88Bacillus gelatini
99 %
Unknown
A,C,D,E, &F %
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.
Evidence of a Novel Species?
Figure 3: Gram stain of unknown 2 showing Gram
positive rods.
Figure 4: Endospore stain of unknown two showing spore
formation
+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
Future ResearchAntimicrobial testing on identified isolates
Focus on unknown 2 Characteristics of unknown 2
morphologyMetabolismIdentification
Acknowledgements Dr. Tammy Tobin Laura Kraker Alex Rebuck Alysha Melynk COB students
Peter Lynch Joneisha MooreRebecca KemmererLauren Kraemer
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