1. Microbial Ecology of the Centralia, Pennsylvania Mine Fire: A Study of
Thermophilic Actinomycetes
Kinley Hardy
Advisor: Dr. Tammy Tobin
Abstract:
An underground mine fire that started in Centralia, Pennsylvania in 1962 has had
a major impact on the soil microbial communities that reside there. Previous research in
this site has documented the presence of novel thermophilic actinomycetes that may be
capable of producing heat-stable antibiotics. In order to isolate and identify additional
novel actinomycetes, soil samples were collected from Centralia at three different
temperatures (32˚C, 48˚C and 65˚C) and inoculated on glycerol yeast agar (fall) or
nutrient agar (spring). Unique colonies were isolated and identified using 16S rRNA gene
sequencing. In the fall, five isolates were identified: Brevivacillus sp., Geobacillus sp.,
Bacillus fumariolo, Streptomyces mexicanus., and Bacillus gelatini. Although a
thermophilic actinomycete was isolated (Streptomyces mexicanus) it is not a novel
species. Geobacillus sp. was identified from an isolate that showed promising
antimicrobial properties in culture and was only a 98% match to a known genus.
Additionally, a metagenomic analysis of the Centralia soil also confirmed the presence of
Geobacillus sp. at 3.5% in 60°C soil. Many species of Geobacillus are the source of a
thermostable antibiotic that has promising potential in food and medicine, suggesting that
this isolate might be a novel species with thermostable antibiotic potential. Further
research will consist of continued soil extraction and antimicrobial testing on previously
identified isolates.
Introduction:

2. Centralia is a small town located in central Pennsylvania. In 1962, a routine trash
fire spread to an open coal seam and started a massive underground fire that is still
burning today. The fire has since reduced the town to rubble, and only a handful of
residents remain. However unfortunate this tragedy is, the Centralia mine fire has become
an important site for scientific research. This is because the fire has changed the entire
ecosystem dramatically (Erickson and White 2008). Steam and gases have vented from
the fire to the surface for decades and have caused significant changes to the temperature
and chemistry of the overlying soils. In particular, the surface soils can reach
temperatures of 200˚C or more, although temperatures ranging from 40˚C to 60˚C are
more common, and these high temperatures have been sustained for long periods of time
(Janzen and Tobin-Janzen 2008). These soil conditions encourage the growth of unique
microbial communities, including thermophilic actinomycetes, soil microbes that thrive
in high temperatures.
Actinomycetes are a heterogeneous group of Gram-positive, aerobic bacteria
(DiSalvo 2010). They are defined as having a high guanine and cytosine base
composition. They also have a characteristic branching, filamentous growth pattern that
results in the formation of a mycelium, and they produce reproductive spores (Gao et. al
2006). These two characteristics often led people to mistake them for fungi before they
were properly identified. Actinomycetes are commonly found in soil where they perform
several critical functions. They are responsible for nitrogen fixation for uptake by plants,
they are environmental decomposers, and some help to maintain the balance in soil
ecosystems with the production of antimicrobials. Actinomycetes also use antimicrobials
for protection by killing off nearby competitors. These antimicrobials are the source of

3. about 50% of today’s commercially produced antibiotics, such as streptomycin and
actinomycin (Waksman et al. 1946). Actinomycetes occur worldwide, and many of them
thrive in hot soil, which is why Centralia, PA is a perfect study site.
Additionally, extreme environments like Centralia have been promising in the
discovery of thermophilic soil microbes that produce secondary metabolites of medical
and industrial importance. Thermophiles with thermostable enzymatic production have
been isolated for potential use in biosynthetic processes, such as biorefinery, and for their
production of thermostable celluloses (Dimise et. al 2007 and Turner et. al 2007).
Preliminary evidence suggests that Centralia’s fire-impacted soils contain
microorganisms that are capable of producing these natural products. Common taxa
include Actinomycetes, Geobacillus, and others such as Acidobacteria and Chloroflexi,
which all have potential for natural product production.
This project seeks to identify novel thermophilic actinomycetes in fire-impacted
soils in Centralia using both culture-based and metagenomic analyses. It is hoped that
these actinomycetes will produce thermostable secondary metablites with antimicrobial
properties.
Methods:
Fall 2013. Surface (1-10 cm depth) soil samples were collected from Centralia
from three different temperatures: 32˚C, 49˚C, and 66˚C. The soil samples were then
placed on ice until arrival at the lab, where they were either used immediately to
inoculate media or were placed into the freezer at -80˚C. The samples to be used for
inoculation were diluted with sterile deionized water in 15mL falcon tubes and vortexed
to break open the soil particles. The dilution was then streaked onto glycerol-yeast agar

4. plates (15g agar, 5g yeast, 50mL glycerol, 950mL DI water). Six isolates were chosen
from those plates (designated unknowns 1-6 in subsequent analyses) based upon unique
morphological characteristics or the presence of a zone of inhibition. These isolates were
first grown on glycerol-yeast agar medium, and then transferred to liquid glycerol-yeast
broth. The broths were allowed to incubate at 55˚C overnight and were checked for
growth daily. Once growth was present, DNA analysis was performed for each isolate
using the MoBio Ultraclean DNA isolation kit. PCR (29 cycles of 95˚C for 4 min, 94˚C
for 1 min, 55˚C for 1 min, 72˚C for 2min, final extension at 72˚C for 5 min) was then
performed using the universal domain Bacteria primers 8F
(GGATCCAGACTTTGATYMTGGCTCAG; Ben-Dov et. al 2006) and 806R
(GGACTACHVGGGTATCTAAT; Walters et. al 2011) to amplify a 798 bp portion of
the 16S rRNA gene in the samples. The presence of a fragment of an appropriate length
was verified using agarose gel electrophoresis, and then the samples were sent to
GenScript to be sequenced. The resulting sequence trace files were quality checked using
4Peaks (http://4peaks.en.softonic.com/mac) and then the sequence files were used to
identify the isolates using the Basic Local Alignment Search Tool (Madden 2002).
Spring 2014. Following BLAST identification and colony analysis of the original
isolates, it was determined that unknown 2 could represent a novel Geobacillus species
with antimicrobial properties, but that none of the other isolates warranted further
investigation. Thus, research efforts were directed in two directions. First, new soil
samples were collected from the same three sites utilized in the fall. These sites showed
slightly different temperatures of 43˚C, 48˚C, and 55˚C. The soil samples were diluted as
previously described but were streaked out on nutrient agar plates (Carolina Biological

5. Supply) in order to foster growth of a wider range of bacteria. Six unique colonies were
chosen for isolation (designated unknowns A-F) using the same criteria as above. The
isolates were identified using 16S rRNA gene sequencing as previously described.
Soil collection and sample preparation for metagenomic analyses. Since most
bacteria currently cannot be cultivated in isolation, the presence of Geobacilli and
actinomycetes in Centralia was also analyzed using culture-independent metagenomic
analysis of the community 16S rRNA genes. For this analysis, genomic DNA was
directly isolated from soil collected in 2012 from 3 boreholes in Centralia, PA (37°C,
52°C and 60°C) using the MoBio PowerSoil DNA Isolation Kit, and sent to the Penn
State Core Genomics Facility for metagenomic analysis. In this facility, PCR with
universal primers 27f (GAGTTTGATCMTGGCTCAG; Lee et. al 2010) and 1492r
(NTACCTTGTTACGACT; Barry et. al 2011) was then used to amplify the community
16S rRNA genes in each of the samples, and pyrosequencing was performed utilizing a
Roche Genome Sequencer FLX.
Analysis of metagenomic data. The resulting sequences were then quality
filtered and analyzed using MacQIIME (Caporaso et. al 2010). Sequences with PHRED
scores below 25, that were not the correct length, or that had too many homopolymers
were not analyzed. The remaining sequences were assigned to operational taxonomic
units (OTUs) based upon sequence similarity, with a 97% and 94% similarity set for
species and genus identity, respectively. A representative sequence from each OTU was
then chosen to represent that OTU for all further analyses. The representative sequences
were compared to all sequences present in the Greengenes 16S rRNA database (DeSantis
et. al 2006) and each OTU identified either as a known taxon, or a potentially new,

6. unassigned taxon. A biom table of the assigned taxonomic info was generated and used to
create bar graphs for better visualization.
The sequences were then aligned and filtered again, removing uninformative data,
in order to create phylogenetic trees. Neighbor-joining trees were generated using
FastTree through the MacQIIME terminal. Topiary Explorer (Pirrung et. al 2011) was
used to visualize and print phylogenetic trees, and to color-code nodes based on sample
metadata.
Results and Discussion:
In the fall, several different bacterial species were identified using 16S rRNA
gene sequencing (Table 1). A potentially novel species of Brevibacillus was isolated from
the 65°C soil. Brevibacillus is a genus of Gram-positive bacteria in the
family Paenibacillaceae. Several different species of these bacteria have been identified
including Brevibacillus brevis, Brevibacillus choshinensis and Brevibacillus fluminis
(Shida et al. 1996). However, since the BLAST analysis showed a 99% match to this
genus, it is likely that our isolate has already been identified.
Bacillus fumarioli has been shown to be a frequent contaminant in gelatin extracts
from the United States and Europe. It was originally isolated from geothermal soils in
Antarctica (De Clerk et al. 2004). In our lab, it was isolated from all three experimental
temperatures, which corroborates the theory from previous literature that this bacterium
has the ability to be versatile and of interest for further study (De Clerk et al. 2004).
Bacillus gelatini is an aerobic, endospore-forming bacterium that has also been shown to
contaminate gelatin (De Clerk et al. 2004). This bacterium was isolated in our lab from
the soil temperatures 32.˚C and 48˚C, suggesting that it grows best at lower soil

7. temperatures and might not be as promising for our research pertaining to thermostable
compounds.
Streptomyces mexicanus is an aerobic, Gram- positive actinomycete that produces
thermostable xylanolytic enzymes. It forms a highly branched substrate mycelium and
aerial hyphae, which can differentiate into several long chains of spores (Petrosyan
2003). The presence of S. mexicanus proves that thermophilic actinomycetes can be
isolated from Centralia soil. However, since unknown 5 was a 99% match to this species,
it is unlikely that it is novel.
Table 1: Table of identified isolates from fall ’13 based upon 16S rRNA gene
sequencing. The temperature each unknown was collected from and the percent identity
to a known genus/species is provided.
Sample ID Temperature
Collected (˚C)
Species with closest
16S rRNA match
% identity
Unknown 1 65 Brevibacillus sp. 99%
Unknown 2 65 Geobacillus sp. 98%
Unknown 3 32, 48 & 65 Bacillus fumariolo 99%
Unknown 5 32 Streptomyces mexicanus 99%
Unknown 6 32 & 48 Bacillus gelatini 99%
Geobacillus is a relatively new genus named by Nazina et al. in 2001. This group
of Russian scientists proposed the new genus while working on isolating thermophilic
bacteria from oilfields near Russia. Through re-analysis of phylogenetic relationships,
they noticed that several species of known Bacilli, as well as a few new isolates they

8. discovered, were much more closely related to each other than to any other Bacilli, thus
creating a new genus which they called Geobacillus (Nazina et al. 2001 and Zeigler
2001).
As shown in Table 1, Unknown 2 was a 98% match to this new genus, indicating
that it was a Geobacillus, but it did not have significant homology at the species level,
suggesting that it could be a new species. Additionally, when unknown 2 was first
cultured in the lab it exhibited a zone of inhibition. As displayed in the figure below
(Figure 1), a zone of inhibition is an area around a central microbe devoid of any
microbial life, meaning that the central microbe is generating an antimicrobial product
(Waksman 1946). Based upon this definition, we can assume that unknown 2 is capable
of producing some form of antimicrobial product.
Geobacilli are Gram-positive obligate
thermophiles. They are aerobic or
facultatively anaerobic and are chemo-
organotrophs. They also produce endospores
and are widely distributed in nature (Ziegler
2001). Unknown 2 displays similar
characteristics to this genus, as seen in
figures 2 and 3 below. Figure 2 shows that
unknown 2 is Gram positive and Figure 3
shows that unknown 2 produces spores. Unknown 2 also optimally grows at 55-60°C in
an aerobic environment. The morphological similarity of unknown 2 to the Geobacillus
genus further suggests that it might be a novel species of Geobacillus.
Figure 1: Image displaying a zone of
inhibition

9. Figure 2: Gram stain of unknown 2 showing Gram-poitive rods.
Figure 3: Endospore stain of unknown 2 showing spore formation.
Additional research reveals that many members of the Geobacillus genus are the
source of a new, promising antibiotic, geobacillin. Geobacillin is an analog to Nisin, a
current commercially produced antibiotic used to combat food-borne pathogens during
food production (Garg 2012). Nisin, and this new antibiotic geobacillin, are classified as
lantibiotics. Lantibiotics are a special class of complex molecules ribosomally

10. synthesized from Gram-positive bacteria that have antibiotic activity (Wiley 2007).
Geobacillin is especially promising as a lantibiotic because it exhibits seven thioether
cross links, two more than nisin, and the most found in any lantibiotic to date. It also
exhibits the same antimicrobial spectrum as nisin, but with increased activity and stability
(Garg 2012). Additionally, since many of the species of the Geobacillus genus are
capable of producing this molecule, and unknown 2 does produce some kind of
antimicrobial, there is a strong possibility that unknown 2 may also produce it or
something similar.
Metagenomic analysis of the Centralia soil also shows the presence of the
Geobacillus genus. Figure 4, below, shows that this genus is present in the highest
temperature soil collected (60˚C) at 3.5%, but not in the 37°C or the 52°C samples. The
presence of this genus at a relatively high percentage in only the hottest soil collected
from Centralia confirms previous research (Nazina et. al 2001; Zeigler 2001) that this
genus requires hot soils to proliferate, and that in hot temperature soils in Centralia, at
least, this genus is a significant member of the soil community. Additionally, the ability
of this genus to live at such high temperatures means that it might be promising in the
quest for thermo-stable products.
Metagenomic analysis also revealed many actinomycetes present in the soil at low
percentages (0.1-1.5%). Members of the genus Streptomyces were found in the lowest
temperature soils, suggesting that this genus grows optimally at slightly elevated, but not
extremely elevated temperatures. In the hottest temperature soil collected (60˚C), other
actinomycetes, such as members of the class Solirubrobacterales were present, further

11. suggesting that Centralia is a prime place to find these potentially novel, understudied
microbes.
Figure 4: Illustrates the relative abundance of each of the microbial 16S rRNA OTUs
identified in soil from borehole 2 (60˚C) according to metagenomic analysis. Larger
bands directly correlate to larger percentages present in the soil. The brown band
indicated with an arrow represents Geobacillus sp. with an abundance of 3.5%. The red
band at the bottom indicates the number of unidentified species, with a relative
abundance of 50%.
A phylogenetic tree was also created with MacQIIME using the Fast Tree
neighbor-joining algorithm. In a subset of that tree shown in figure 5, below, the
Geobacillus genus is present again only in the 60˚C soil (green branch) and no other
Firmicutes are present in this soil sample. Geobacillus clusters most closely in this
sample with members of the phylum Acidobacteria. This phylum contains soil bacteria
that are particularly adept at surviving in extreme conditions. Interestingly, genome
sequencing of members of the two families most closely related to unknown 2,

12. Solibacteraceae and Koribacteraceae shows that while they are heterotrophs, they are
also able to utilize CO as a carbon source and nitrate as a final electron acceptor in
respiration, both of which are plentiful in Centralia as a result of the fire. Polyketide
synthase genes are also present in these bacteria, and they are known producers of
bacterial cellulose. Thus, future studies of Centralia Acidobacteria could also result in
the discovery of new bioproducts.
Figure 5: Section of a 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 (red 37˚C, blue 52˚C, green 60˚C). The branch highlighted is Geobacillus
sp.
Conclusion:
Several different isolates were successfully identified from Centralia soil, but only
one thermophilic actinomycete was identified and it was not a novel species. However, a
potentially novel species of Geobacillus was also identified, and it has promising

13. qualities for future antibiotic production. Further research will be done on identified
isolates to determine potential antimicrobial properties and new soil samples will be
extracted from Centralia for continued research.
References:
Ben-Dov E, Shapiro OH, Siboni N, Kushmaro A. (2006). Advantage of using inosine at
the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity.
Appl. Environ. Microbiol. 72, 6902-6906.
Berry D, Mahfoudh KB, Wagner M, Loy A. (2011). Barcoded primers used in multiplex
amplicon pyrosequencing bias amplification. Appl. Environ. Microbiol. 77, 7846-9.
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer
N, Gonzalez Pena A, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D,
Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J,
Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight
R. (2010). QIIME allows analysis of high-throughput community sequencing data.
Nature Methods 7(5): 335-336.
De Clerck, E., Gevers, D., Sergeant, K., Rodrı́guez-Dı́az, M., Herman, L., Logan, N. A.,
& De Vos, P. (2004). Genomic and phenotypic comparison of Bacillus fumarioli isolates
from geothermal Antarctic soil and gelatine. Research in microbiology, 155(6), 483-490.
De Clerck, E., Rodriguez-Diaz, M., Vanhoutte, T., Heyrman, J., Logan, N. A., & De Vos,
P. (2004). Anoxybacillus contaminans sp. nov. and Bacillus gelatini sp. nov., isolated
from contaminated gelatin batches. International journal of systematic and evolutionary
microbiology, 54(3), 941-946.
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. (2006).
Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible
with ARB. Appl Environ Microb 72(7): 5069-5072.
Dimise, E. J., Widboom, P. F., Bruner, S. D. (2008). Structure elucidation and
biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile
Thermobifida fusca. Proc. Natl. Acad. Sci. U. S. A. 105, 15311–15316.
DiSalvo, A. “Mycology - Chapter Two Actinomycetes.” (2010).Microbiology and
Immunology Online University of South Carolina School of Medicine. Web. 2
December, 2013. <http://pathmicro.med.sc.edu/mycology/mycology-2.htm>
Erickson, H. E., & White, R. (2008). Soils under fire: soils research and the Joint Fire
Science Program.

14. Gao, B., Paramanathan, R., & Gupta, R. S. (2006). Signature proteins that are distinctive
characteristics of Actinobacteria and their subgroups. Antonie van Leeuwenhoek, 90(1),
69-91.
Garg, N., Tang, W., Goto, Y., Nair, S. K., & van der Donk, W. A. (2012). Lantibiotics
from Geobacillus thermodenitrificans. Proceedings of the National Academy of Sciences,
109(14), 5241-5246.
Janzen, Chris and Tammy Tobin-Janzen. (2008). Microbial communities is fire-affected
soils. Soil Biology, 13: 299-316.
Lee TK, Van Doan T, Yoo K, Choi S, Kim C, Park J. (2010). Discovery of commonly
existing anode biofilm microbes in two different wastewater treatment MFCs using FLX
Titanium pyrosequencing. Appl. Microbiol. Biotechnol. 87, 2335-2343
Madden T. The BLAST Sequence Analysis Tool. (2002). Oct 9 [Updated 2003 Aug 13].
In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD):
National Center for Biotechnology Information (US); 2002-. Chapter 16. Available from:
http://www.ncbi.nlm.nih.gov/books/NBK21097/
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
bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus
uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus,
Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus
thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G.
th.International Journal of Systematic and Evolutionary Microbiology, 51(2), 433-446.
Petrosyan, P., García-Varela, M., Luz-Madrigal, A., Huitrón, C., & Flores, M. E. (2003).
Streptomyces mexicanus sp. nov., a xylanolytic micro-organism isolated from soil.
International journal of systematic and evolutionary microbiology, 53(1), 269-273.
Pirrung, Meg, Ryan Kennedy, J. Gregory Caporaso, Jesse Stombaugh, Doug Wendel, and
Rob Knight. TopiaryExplorer: An application for connecting large phylogenetic trees to
environmental metadata (Bioinformatics 2011).
Shida, O., Takagi, H., Kadowaki, K., & Komagata, K. (1996). Proposal for Two New
Genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov.International journal of
systematic bacteriology, 46(4), 939-946.
Turner, P., Mamo, G., Karlsson, E. N. (2007). Potential and utilization of thermophiles
and thermostable enzymes in biorefining. Microb. Cell Fact. 6, 9.
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.

15. Walters WA, Caporaso JG, Lauber CL,Berg-Lyons D, Fierer N, Knight R. (2011).
PrimerProspector: de novo design and taxonomic analysis of barcoded PCR primers.
Bioinformatics. 10.1093/bioinformatics/btr087.
Willey, J. M., & van Der Donk, W. A. (2007). Lantibiotics: peptides of diverse structure
and function. Annu. Rev. Microbiol., 61, 477-501.
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
4Peaks. http://4peaks.en.softonic.com/mac