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

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

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