MICROBIAL DIVERSITY IN HAWAIIAN FUMAROLES
_______________
A Thesis
Presented to the
Faculty of
San Diego State University
_______________
In Partial Fulfillment
of the Requirements for the Degree
Master of Science in Biology
with a Concentration in
Molecular Biology
_______________
by
Katherine M. Wall
Summer 2011
iii
Copyright © 2011
by
Katherine M. Wall
All Rights Reserved
iv
ABSTRACT OF THE THESIS
Microbial Diversity in Hawaiian Fumaroles
by
Katherine M. Wall
Master of Science in Biology
San Diego State University, 2011
Fumaroles, though little studied, have previously been shown to harbor novel lineages
of microorganisms. In this study, sediments were collected from inside Hawaiian fumaroles
and analyzed using culture independent methods. We were able to extract environmental
DNAs and amplify both Bacterial and Archaeal DNA from 5 locations across Hawaii.
Microscopic examination of the sediments revealed organisms stained for dsDNA with
DAPI. Analysis of the bacterial DNA revealed many sequences similar to uncultured
Cyanobacteria, as well as uncultured lithotrophs and a number of sequences for which the
metabolism was unknown. Archaeal sequences were all from the Crenarchaeota, and
diversity was low. Unifrac analysis of the bacterial sequences showed that the microbial
communities of the fumaroles studied were very similar to each other.
v
TABLE OF CONTENTS
PAGE
ABSTRACT............................................................................................................................. iv
LIST OF TABLES................................................................................................................... vi
LIST OF FIGURES ................................................................................................................ vii
CHAPTER
1
INTRODUCTION .........................................................................................................1
2
MATERIALS AND METHODS...................................................................................3
Collection of Steam and Steam Sediments ..............................................................3
Microscopy ..............................................................................................................4
DNA Extraction and PCR........................................................................................4
Cloning and RFLP Analysis ....................................................................................5
Phylogenetic Analysis..............................................................................................5
3
RESULTS AND DISCUSSION ....................................................................................7
ACKNOWLEDGEMENTS...................................................................................................222
REFERENCES ........................................................................................................................22
vi
LIST OF TABLES
PAGE
Table 1. Specimen Collection Locations and Conditions..........................................................8
Table 2. Identification of the 16s rRNA Bacterial Cloned Sequences Using NCBI
Taxonomy ....................................................................................................................11
Table 3. Identification of the 16s rRNA Archaeal Cloned Sequences Using NCBI
Taxonomy ....................................................................................................................17
vii
LIST OF FIGURES
PAGE
Figure 1. Images of steam vents in Hawaii................................................................................9
Figure 2. DAPI stained micrographs of steam vent sediments................................................10
Figure 3. Phylogenetic tree showing the relationships of the steam vent bacterial
sequences with their near neighbors. ...........................................................................13
Figure 4. Phylogenetic tree showing Archaeal sequences from steam vents and near
neighbor sequences downloaded from RDP. ...............................................................15
Figure 5. Unifrac PCA analysis shows how the different environments are related to
each other. ....................................................................................................................18
Figure 6. Unifrac environment distance matrix and P-test significance. The Unifrac
distance matrix computes distances between environments........................................19
1
CHAPTER 1
INTRODUCTION
Fumaroles (aka. geothermal steam vents), are formed when rainwater is heated by
magma and is re-emitted as steam, venting through volcanic deposits. Fumarole steam may
also be mixed with volcanic gases, such as CO2, SO2, and H2S (4). Fumaroles and
geothermal soils contain little organic carbon or nitrogen, but are rich in minerals, and thus
can provide an energy source for lithotrophic organisms (9). The presence of abundant water
from the condensed steam further enables organisms to live in these environments.
Despite decades of research in geothermal ecosystems, fumarole associated microbial
communities have received little attention. A partial explanation for the paucity of research
lies in the difficulty of extracting DNA from fumarole sediments (19). Volcanic soils such as
fumarole sediments can be acidic and also may contain minerals such as magnesium, both of
which impede DNA isolation and downstream applications such as PCR (1, 7). Recently a
few labs, including ours, have successfully managed to extract significant DNA for culture
independent molecular analysis of Bacterial (1) and Archaeal (1, 6) fumarole communities.
These studies have uncovered highly diverse and complex communities, suggesting that
extreme environments may select for deeply divergent and unusual organisms which makes
them worthy of studying, despite the difficulties.
One study that examined fumarole associated microbial communities was performed
on the fumaroles of the Canary Islands (14). This study focused on the question of
colonization and immigration of new microbes into the volcanic environments. They found
that immigration of new organisms was occurring continuously. A study on fumarole
microbial diversity and their environments was also done in the Galapagos Islands, a
geographically isolated group of islands (11). The fumaroles that were studied varied in
factors such as pH, temperature, and chemical composition. They found that the microbial
communities clustered according to pH. Because pH depends on the chemistry of the
substrate, the chemistry undoubtedly had an effect on the microbial diversity. Another study
on fumarole microbes was done at the Socompa Volcano in the Andes, on the border
2
between Argentina and Peru (3). Socompa volcano hosts unique microbial mats that are
associated with the fumaroles on the volcano. Species in this community were found to be
closely related to easily dispersed organisms.
In Hawaii, the focus of the present study, there have been numerous studies of
microbes in volcanic soils, but only two on fumarole associated microbes. Ellis et al. (2008)
found somewhat surprisingly that condensed steam from Hawaiian steam vents contained
halophilic Archaea (6). A second study by Benson et al., (2010) was the first to find Archaea
in fumarole vent sediments. In that study, the steam sediments were found to contain novel
lineages of chemolithotrophic Crenarchaeota related to ammonia oxidizers found commonly
in marine habitats (1).
Clearly much more research needs to be done to fully understand microbial diversity
and the processes that shape it. In this study, we pursued a deeper and more comprehensive
investigation of the microbial communities associated with Hawaiian steam vent sediments.
This involved a much wider sampling of vents across the big island and analysis of both
bacterial and archaeal communities in steam sediments. In this paper, we utilized culture
independent analyses to investigate the microbial communities present in Hawaiian fumarole
sediments. We found that the microbial communities living in the vents were dominated by
photosynthetic organisms, with a smaller assemblage of lithotrophs and microbes of
unknown metabolism. The microbial communities inside the vents were found to be similar
to each other, and did not cluster along any environmental axis (such as pH, temperature, or
sediment type).
3
CHAPTER 2
MATERIALS AND METHODS
COLLECTION OF STEAM AND STEAM SEDIMENTS
The main island of Hawaii is the location of the active volcanoes in the island chain
of Hawaii. The steam vents are primarily located in Hawaii Volcanoes National Park, with
some steam vents located outside of the park. Due to the HVNP sampling permit, we are
unable to disclose the sampling locations, and locations are given as codes. One exception is
Pahoa Steam Caves, which is outside the park. The steam vents vary in temperature, pH, and
contain several types of sediment. Sediment types were generally classified into iron
containing sediment (red or brown in color), white crystalline material (collected from inside
the vents) and sulfurous sediments.
In this study we collected sediments and steam from 5 different locations. At each
location several vents (minimum of 2, up to 5 in some locations) were targeted for collection.
Sediments were collected from the walls and roofs of the vents and in some cases from
deposits around the outside of the vent. In all cases the areas from which sediment was
collected were in continuous contact with the steam. During collection, temperature and pH
readings of the steam were taken. pH readings were made using condensed steam.
Sediments inside the vents were collected with a sterile 50 ml conical plastic tube
attached to a pole. The edge of the plastic tube was scraped against the vent surface, and the
sediment fell in the tube and was collected. To minimize soil contamination, a thin layer of
sediment 0.5 cm – 1 cm from the surface of the vent was collected. Tubes were capped
immediately after collection and labeled. Tubes with sediments were kept at ambient
temperature during transport to the lab. Samples for chemical analysis and culture remained
at ambient temperature, while sample portions destined for DNA extraction were frozen at 20C.
4
MICROSCOPY
To image cells, between 0.05 and 0.1 g of sediment (estimated) was placed in a sterile
2 ml tube, and 0.1 ml of sterile PBS pH 7.4 was added. The tube was vortexed to mix the
sediment, and 30 μl of the suspended sediment/PBS mixture was transferred to a clean tube.
3 μl of a 1:100 dilution of 1mg/ml DAPI stock solution was added, and the sediment
suspension was stained for 10 minutes in the dark. The suspension was then centrifuged at
10K RPM for 2 minutes, and the fluid removed. 15 μl of sterile PBS pH 7.4 was then added
to the tube and mixed. This suspension was observed on a Zeiss Axio Observer DI and
photographed with an attached Zeiss MRc camera (Zeiss, Oberkochen, Germany) and
Axiovision software (Zeiss). Images were adjusted for contrast and brightness using
GraphicConverter.
DNA EXTRACTION AND PCR
Genomic DNAs were extracted from the sediments using the PowerSoil DNA
Isolation kit (MO BIO Laboratories). Between 0.2 and 0.5 g of sediment was weighed out
aseptically in a laminar flow hood and extracted precisely following the kit’s supplied
protocol. Negative controls (sediment free) were also performed each time samples were
processed. These controls were carried through subsequent PCR steps.
For each extracted DNA sample, 16S rRNA gene sequences were amplified with
Bacteria specific and Archaea specific primers. The primers used for archaeal DNA
amplification were 21F[5'-TCCGGTTGATCCYGCCGG-3] (5) and 915R [5’GTGCTGCCCCGCCAATTCCT- 3’] (17), and for bacteria 27F [5'AGAGTTTGATCCTGGCTCAG-3'](17) and 805R [5'-AGAGTTTGATCCTGGCTCAG-3']
(20) primers were used. PCR reactions were performed in 100 μl, each of which included: 1X
Sigma PCR buffer without MgCl2, 2mM MgCl2, 0.3μM of each primer, 0.2 mg/ml BSA and
5 unites of Taq DNA polymerase. For each PCR reaction a negative control containing the
reaction mix but no DNA template, and a positive control with either bacterial DNA or
archaeal DNA, was run in parallel with the other samples. In addition to the PCR controls,
the DNA extraction negative extraction control (no sediment added) was used as template for
PCR reaction to check for contamination.
5
Thermocycler parameters included an initial denaturing step of 10 minutes at 95°C,
followed by 35 cycles of: 1.5 minutes at 95°C, 1 minute annealing at 55°C, 1.5 minute
extension step at 72°C, a 20 minute final extension step at 72°C, and held at 4°C until
removed (Eppendorf Mastercycler Gradient thermocycler). PCR amplified DNA was run on
an agarose gel (1% agarose/TAE for 0.5 hr at 130V) to check for bands. Positive PCR
reactions selected for cloning were purified using the Qiaquick PCR cleanup kit, or gel
purified using a 2% agarose gel and the Qiaquick gel purification kit. Purified DNA was
quantified using a Nanodrop spectrophotometer.
CLONING AND RFLP ANALYSIS
Cloning of amplified 16S gene DNA was performed using a TOPO-TA (Invitrogen)
cloning kit and following the manufacturers instructions. Between 12 and 60 positive clones
were picked for each reaction, grown overnight in selective broth, and screened via PCR for
inserts using M13F and M13R primers. PCR products were assessed for positive clones by
running on a 1% agarose gel (1% agarose/TAE for 0.5 hr at 130V).
To screen for sequence variability, the M13 amplified PCR products were digested
with a cocktail of three enzymes. Enzymes used were Not 1, EcoR1, and AVA II (Fermentas)
The enzymes were diluted to 2x in 2x Tango Buffer, mixed 1:1 with PCR product, and
incubated for 1 hour at 37°C. Digests were then run out on a 2% low-melt agarose gel and
analyzed. Clones with unique banding patterns were sent to Eton Biosciences for sequencing
using the M13 primers.
PHYLOGENETIC ANALYSIS
Sequence chromatograms were checked for quality and trimmed manually. The
sequences were also checked for vector contamination using the NCBI Vecscreen tool and
any contaminating vector was excised. Contigs were made using the CodonCode Aligner, for
clones which were sequenced in both directions. For sequences that would not contig (due to
insufficient length of high quality sequence) or were not sequenced in both directions, the
forward direction sequence was used for analysis. Sequences were aligned using the LBL
Greengenes aligner (http://greengenes.lbl.gov/cgi-bin/nph-NAST_align.cgi), and uploaded to
RDP (http://rdp.cme.msu.edu/classifier/classifier.jsp) for classification using their Bayesian
6
rRNA classifier (2). Sequences were also BLASTed manually using the NCBI BLAST
interface (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Nucleotides). Any sequences
that showed evidence of chimeras based on the NCBI blast results were excluded from
further analysis.
Nearest neighbor sequences were downloaded from RDP, aligned using the LBL
Greengenes aligner, converted to Phylip format using Readseq (http://www.ebi.ac.uk/cgibin/readseq.cgi) and uploaded to the UCSD Cipres Science Gateway (12). Phylogenetic trees
were then constructed using the RAxML Black Box program (18). Both cultured and
uncultured near neighbor sequences were included in phylogenetic trees. In addition to
RAxML Black Box, MrBayes was also used to construct phylogenetic trees, and the results
not reported. The RAxML Black Box bootstrapping values and MrBayes posterior
probability values were reported.
For Unifrac analysis, RAxML Black Box phylogenetic trees of the cloned bacterial
sequences were created (using the methods previously described) and uploaded to Unifrac
(10), along with an environment file. The environments described in the file were based on
the location of the steam vents, the temperature, the pH, and the appearance of the sediment
(red or dark brown iron containing sediment, white crystals, or sulfur containing sediment).
Included in the environment file were the counts of each sequence, which were obtained
from the RFLP banding patterns.
7
CHAPTER 3
RESULTS AND DISCUSSION
Table 1 details the vents sampled and the results of DNA extraction and PCR efforts,
and Figure 1 shows representative vents from the various locations. It was difficult to
visualize microorganisms in the steam vent sediments, due to autofluorescence of the
sediments. Organisms that could be seen were typically found attached to sediment particles
and coccoid in shape (Figure 2). 15 out of the 21 samples tested yielded sufficient DNA for
PCR analysis. We were able to amplify 16S rRNA gene sequence from 10 of those samples,
and 8 samples were positive for archaeal 16S rRNA. The negative DNA extraction controls
and the negative PCR controls yielded no PCR bands in every instance. All the positive PCR
reactions were successfully cloned and yielded positive clones for analysis.
Blast analysis found that the majority of the sequences determined from these vents
matched uncultured organisms (Table 2). The closest matches tended to be sequences
isolated from geothermal environments, especially geothermally heated volcanic soils and
hot springs. However we also found sequences closely related to Bacteria and Archaea from
a wide variety of environments, including Hawaiian volcanic deposits (non-geothermal),
various soils, and mines (Table 2, Figure 3 and Figure 4).
Bacterial sequences from Location 1 were dominated by Cyanobacteria,
compromising the majority of sequences in this location. In addition to Cyanobacteria, there
were several members of the Chloroflexi (green non-sulfur bacteria) group, suggesting that
photosynthesis was the most dominant metabolism present. One other autotroph was
identified and this was most similar to an Acidophilium strain, a chemolithotroph. One
sequence was found to be similar to a known heterotroph, Ktedobacter racemifer. Other
sequences were not similar to any organisms with a known metabolism so it was not possible
to draw any conclusions about their metabolism.
8
Table 1. Specimen Collection Locations and Conditions
Temp(C)
pH
Type
Bacterial DNA Archaeal DNA
Location 1
Vent 1
Vent 2
Vent 3
Vent 4
Vent 5
40
41
70
ND
76
4.5
4.5
5.3
ND
6
Non-Sulfur
Non-Sulfur
Non-Sulfur
Non-Sulfur
Non-Sulfur
+
+
+
-
+
-
Location 2
Vent 1
Vent 2
Vent 3
Vent 4
Vent 5
65
77
55
68
25
5
5.5
5.5
4.8
4.8
Non-Sulfur
Non-Sulfur
Non-Sulfur
Non-Sulfur
Non-Sulfur
+
+
-
+
-
Location 3
Vent 1
Vent 2
60
66
5.5
5.5
Non-Sulfur
Non-Sulfur
+
+
+
+
Location 4
Vent 1
Vent 2
Vent 3
66
71
77
5
5
5
Non-Sulfur
Non-Sulfur
Non-Sulfur
-
-
Location 5
Vent 1
Vent 2
Vent 3
Vent 4
80
93
90
49
5
ND
5
ND
Sulfur
Sulfur
Sulfur
Sulfur
ND
ND
ND
ND
ND
ND
ND
ND
* ND means the measurement was not taken.
9
Figure 1. Images of steam vents in Hawaii. (A) Location 1 Vent, showing a raised
section of lava with steam issuing from the end of the raised region. (B) Location 2,
showing steam issuing from cracks in the lava. (C) Pahoa Steam Caves (Location 3) a
small partially collapsed lava bubble with steam coming out of the top. (D) Location 4,
showing a steam vent open to the elements with vegetation surrounding it. (E)
Location 5, the only sulfurous steam vents in the study. Sulfur has precipitated out of
the fumarolic gas and collected on the edges of the vent. (F) A closeup of a vent from
Location 1 showing white crystalline material inside the vent.
10
Figure 2. DAPI stained micrographs of steam vent sediments. On the left is the
combined brightfield and DAPI channel images, on the right is the DAPI channel
alone. Scale bars are 10 M. (A) Location 2 sediment (B) Location 3 (Pahoa steam
caves sediment) (C) Location 1 sediment.
KWMUSI517
KWMUVI424
KWMUFK561
KWMUFK559
KWMUFK563
KWMUTI493
KWMUVI428
KWMUSI518
KWMUVI421
KWMUVI426
KWMUVI427
KWMUFK558
KWMUFK560
KWMUFK562
KWMUTI494
KWMUTI495
KWMUTI496
Location 2
KWKI00K81
KWKIEK605
KWKIEK679
KWKIEK603
KWKIEK612
KWKIEK601
KWKIEK609
KWKIEK684
KWKIEK685
KWKIEK610
KWKIEK602
KWKIEK604
KWKIEK606
KWKIEK608
KWKIEK611
KWKIEK678
KWKIEK680
KWKIEK683
KWKIEK687
Location 1
Clone ID
Chlorogloeopsis sp. ART2B_179
Chlorogloeopsis sp. Greenland_2
Uncultured bacterium clone ESCHR-1
Chlorogloeopsis sp. Greenland_2
Uncultured bacterium clone
Meiothermus silvanus DSM 9946
Acidiphilium sp. BGR 71
Uncultured bacterium clone B26
Uncultured Fibrobacteres/Acidobacteria
Uncultured bacterium clone
Roseiflexus sp. RS-1
Xanthomonadaceae bacterium
Chloroflexus aggregans DSM 9485
Meiothermus silvanus DSM 9946
Ktedobacter racemifer
Uncultured bacterium clone FFCH13977
Meiothermus silvanus DSM 9946
Uncultured bacterium clone 1969b-30
Uncultured bacterium clone S5-20
NCBI Blast Result
Proteobacteria
Uncultured Limnobacter
Cyanobacteria
Chlorogloeopsis sp. Greenland_2
Cyanobacteria
Chlorogloeopsis sp. Greenland_2
Chloroflexi
Uncultured bacterium clone B21
Cyanobacteria
Uncultured Chlorogloeopsis sp. Clone
Cyanobacteria
Uncultured Chlorogloeopsis sp. ART2B_179
Cyanobacteria
Chlorogloeopsis sp. Greenland_5
Proteobacteria
Sediminibacterium sp. nju-T3
Bacteriodetes
Chitinophagaceae bacterium
Firmicutes
Candidate division OP10 bacterium
Cyanobacteria
Uncultured bacterium
Chloroflexi
Uncultured bacterium clone TAb-38
Cyanobacteria
Uncultured Chlorogloeopsis sp. Greenland_2
Cyanobacteria
Uncultured bacterium clone FCPS406
Cyanobacteria
Uncultured bacterium clone L11C27HI1NSC
Thermodesulfobacteria Uncultured bacterium clone HDB_SIPO614
Actinobacteria
Uncultured bacterium clone TCb-48
Cyanobacteria
Cyanobacteria
Firmicutes
Cyanobacteria
Cyanobacteria
Thermi
Proteobacteria
Actinobacteria
Chloroflexi
Actinobacteria
Chloroflexi
Proteobacteria
Chloroflexi
Thermi
Proteobacteria
Cyanobacteria
Proteobacteria
Cyanobacteria
Cyanobacteria
Phylum
FJ804448.1
DQ430997.1
DQ430997.1
FJ466084.1
JF303684.1
JF303684.1
DQ431000.1
FJ915158.1
FN665661.1
AM749780.1
DQ791287.1
DQ791314.1
DQ430997.1
EF516646.1
GU292506.1
HM187162.1
DQ791460.1
JF303684.1
DQ430997.1
EU863591.1
DQ430997.1
FJ569585.1
CP002042.1
GU167994.1
FJ466013.1
AY387376.1
AY917751.1
CP000686.1
FR774560.1
CP001337.1
CP002042.1
AM180156.1
EU134645.1
CP002042.1
AY917751.1
EU680443.1
Accession No.
97%
99%
99%
98%
99%
97%
100%
93%
100%
97%
91%
88%
99%
87%
95%
87%
99%
99%
99%
97%
99%
87%
91%
97%
91%
88%
97%
87%
98%
80%
91%
97%
94%
96%
97%
86%
%Identity
uranium mine, India
Greenland hot spring
Greenland hot spring
Kilauea volcanic deposit
Yellowstone stromatalite
Yellowstone stromatolite
Greenland hot spring
mine tailings, China
water sample
steam heated soil
Kilauea volcanic deposit
Kilauea volcanic deposit
Greenland hot spring
soil
geothermal steam vent
radiowaste contaminated soil
Kilauea volcanic deposit
Yellowstone stromatolite
Greenland hot spring
chrysanthemum thrip
Greenland hot spring
alpine tundra soil
Portugal hot spring
sulfidic mine waste
Hawaiian volcanic deposit
tropical rainforest soil
Hawaii volcanic deposit
Octopus spring, Yellowstone
paper machine bacteria
neutral to alkaline hot spring
Greenland hot spring
soil
tallgrass prairie soil
portugal hot spring
Hawaiian volcanic deposit
forest soil, China
Environment
Table 2. Identification of the 16s rRNA Bacterial Cloned Sequences Using NCBI Taxonomy
5
3
3
2
2
2
2
1
1
1
1
1
1
1
1
1
1
28
9
3
7
3
2
2
2
2
2
2
1
1
1
1
1
1
1
1
Counts
(table continues)
11
Proteobactera
Firmicutes
Proteobactera
Actinobacteria
Firmicutes
Firmicutes
Proteobactera
Aquificae
Bacteriodete
Cyanobacteria
Cyanobacteria
Chloroflexi
Firmicutes
Proteobacteria
Actinobacteria
Thermogagae
Proteobacteria
Proteobacteria
Firmicutes
Gemmatimonadetes
unclassified
Actinobacteria
Actinobacteria
Actinobacteria
Thermogagae
Bacteriodetes
Chloroflexi
Thermogagae
Chloroflexi
Thermi
Phylum
Sulfobacillus acidophilus strain DK-I15/45
Sulfobacillus thermosulfidooxidans
Acidithiobacillus caldus
Kocuria rhizophila
Sulfobacillus acidophilus strain DK-I15/45
Uncultured bacterium clone LY-43 16S
Micrococcus sp. WB20-02
Uncultured bacterium clone TCb-48
Uncultured Bacteroidetes bacterium
Chlorogloeopsis sp. Greenland_2
Thermogemmatispora foliorum
Roseiflexus castenholzii DSM 13941
Uncultured bacterium
Uncultured bacterium clone R15
Uncultured bacterium clone L11C27HI1NSC
Uncultured firmicute clone SM2D03
Uncultured bacterium clone
Uncultured Bacteroidetes
Uncultured bacterium clone TCa-11
Uncultured bacterium clone HDB_S
Uncultured bacterium clone BG225
Uncultured bacterium clone BG225
Uncultured bacterium clone TCa-11
Uncultured bacterium HDB_SIPC476
Uncultured bacterium clone TCc-09
Uncultured bacterium clone 10D-3
Thermogemmatispora foliorum
Uncultured bacterium clone L11C30HI1NSC
Uncultured bacterium clone N707B_334
Uncultured bacterium clone BG225
NCBI Blast
EU419196.1
AB089844.1
X72851.1
AP009152.1
EU419196.1
JF429148.1
GU595337.1
DQ791460.1
FN666226.1
DQ430997.1
AB547913.1
CP000804.1
FN659201.1
AF407687.1
GU292506.1
AF445720.1
HM362606.1
AB113613.1
DQ791400.1
HM187162.1
HM362606.1
HM362606.1
DQ791400.1
HM186891.1
DQ791461.1
DQ906856.1
AB547913.1
GU292508.1
GU941113.1
HM362606.1
Accession No.
94%
99%
100%
99%
99%
99%
99%
97%
98%
99%
84%
86%
91%
99%
96%
96%
98%
96%
98%
93%
99%
97%
89%
84%
99%
84%
84%
93%
99%
98%
%Identity
hydrothermal volcanic soil
acid mine drainage
low nutrient soil
hydrothermal volcanic soil
Kilauea volcanic deposit
Tunisian hot spring
Arctic hot spring
geothermal soil, Japan
Japanese hot spring
earthworm gut
geothermal artesian water
geothermal steam vent
geothermal soil, Yellowstone
compost pile
geothermal mine stream
Kilauea volcanic deposit
radiowaste contaminated soil
compost
compost
Kilauea volcanic deposit
radiowaste contaminated soil
Kilauea volcanic deposit
subsurface soil
geothermal soil, Japan
geothermal steam vent
South China Sea
compost pile
Environment
4
1
1
1
1
1
1
6
4
3
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Counts
Percent identity and environment from which the most similar sequences were isolated are shown, as well as the counts of each
sequence.
*The
KWSF00089
KWSF00013
KWSF00014
KWSF00015
KWSF00090
KWSF00091
KWSF00092
Location 5
KWPAFI530
KWPAFI549
KWPAFI550
KWPAFI531
KWPAFI547
KWPAFI548
KWPAFI532
KWPASI599
KWPAFI533
KWPAFI534
KWPAFI537
KWPAFI538
KWPAFI539
KWPAFI540
KWPAFI541
KWPAFI542
KWPAFI543
KWPAFI544
KWPAFI545
KWPAFI551
KWPASI598
KWPASI6101
KWPASI6102
Location 3
Clone ID
Table 2. (continued)
12
13
Figure 3. Phylogenetic tree showing the relationships of the steam vent bacterial
sequences with their near neighbors. Nearest neighbor sequences were downloaded
from RDP. Trees were created with RAxML Black Box and MrBayes. Maximum
likelihood bootstrap values and Bayesian probabilities are shown. Bootstrap values are
shown on the left, and MrBayes probabilities are shown on the right.
14
15
Figure 4.. Phylogenetic tre
treee showing Archaeal sequences from steam vents and near
neighbor sequences downloaded from RDP. Tree was constructed using RAxML
Black Box and MrBayes. RAxML bootstrap values are shown on the left, and
MrBayes probability values shown on the left
left.
16
Location 2 was similarly dominated by Cyanobacteria, however the most abundant
microbe has similarity to an organism found in uranium mine. This bacterium is member of
the Limnobacter group, which are chemolithoheterotrophs and can oxidize thiosulfate to
sulfate (16). In addition, there was one member of the Thermodesulfobacteria group present,
which are sulfate reducing bacteria that can also oxidize organic substrates such as acetate
(8). Unlike Locations 1 and 2, Location 3 was somewhat different in its bacterial composition
in that it was not dominated by Cyanobacteria. In this case, the most abundant organism were
members of Aquificales, a group consisting of chemolithotrophic thermophiles. There were
also several Chloroflexi present, and together with the Cyanobacteria the photosynthesizers
outnumbered the chemolithotrophs in this location as well.
Location 5 was the only location that contained large amounts of sulfur, both
elemental sulfur substrate and sulfur containing gases, and this was reflected in the biota
present. Organisms most similar to Sulfobacillus acidophilus were the most abundant
sequences found, with other members of Sulfobacillus such as Sulfobacillus
thermosulfooxidans present. Sulfobacillus members are mixotrophs: they can grow
lithotrophically on ferrous iron and mineral sulfides, and can also grow as heterotrophs (13).
We found one sequence similar to Acidithiobacillus caldus, which is an obligate lithotroph
that oxidizes sulfur (15).
Consistent with previous studies, we were only able to amplify archaeal sequences
from a small number of vent sediments. Whether this is because the Archaea in these
environments are resistant to our extraction methods or are present at low abundances is
currently unknown. Archaea were identified from a total of 4 environments but diversity was
limited compared to the Bacteria from the same vents (Table 3). All four environments
yielded solely Crenarcheaotes, a finding that was previously observed by Courtney Benson
(1). Most of the Archaea were most similar to uncultured organisms, making it difficult to
draw conclusions about their metabolism and their role in the communities present in the
steam vents. Interestingly, several clones were very closely related to Archaea from mines,
which suggests a deep subsurface origin (Figure 4). The exception was Sulfolobus islandicus,
a heterotroph that was present in two locations with high sequence identity.
Results from Unifrac analysis are shown in Figures 5 and 6. In Figure 5, the
environment distance matrix gives distances between each environment, and the p-test data
Crenarchaeota
Crenarchaeota
Crenarchaeota
Crenarchaeota
Crenarchaeota
Crenarchaeota
Crenarchaeota
Crenarchaeota
Crenarchaeota
Location 1
KWKIEK653
KWKIEK655
KWKIEK654
KWKIEK656
Location 2
KWMUVI65
Location 3
KWPAFI546
KWPAFI536
KWPAFI535
Location 5
KWSF00066
Sulfolobus islandicus HVE10/4
Uncultured archaeon clone 405
Uncultured archaeon clone
Uncultured archaeon
Sulfolobus islandicus HVE10/4
Uncultured archaeon clone kaa142
Uncultured archaeon clone Ta1-a30
Uncultured archaeon clone Ta1-a30
Uncultured archaeon clone kaa142
NCBI Blast
CP002426.1
FJ821635.1
GU221921.1
AB302038.1
CP002426.1
FJ936705.1
DQ791489.1
DQ791489.1
FJ936705.1
Accession No.
100%
99%
97%
95%
100%
98%
99%
99%
98%
%Identity
icelandic hot spring
hot spring sediment
geothermal steam vent
hydrothermal field, Okinawa
icelandic hot spring
Kamtchatka volcanic mud
Kilauea geothermal soil
Kilauea geothermal soil
Kamtchatka volcanic mud
Environment
*The percent identity and environment from which the most similar sequences were isolated are shown.
Phylum
Clone ID
Table 3. Identification of the 16s rRNA Archaeal Cloned Sequences Using NCBI Taxonomy
30
12
6
2
8
4
2
1
1
Counts
17
18
Figure 5. Unifrac PCA analysis shows how the different environments are
related to each other. None of the factors that we investigated explained the
variation between the environments.
19
Unifrac Environment Distance Matrix
P-Test Significance
Figure 6. Unifrac environment distance matrix and P-test significance. The
Unifrac distance matrix computes distances between environments. Pink
highlighted cells indicate the least distance between environments, while blue
highlighted cells indicate the greatest distance between environments. The P-Test
significance computes the significance of distances between environments. In this
P-Test the distances between the environments are not significant.
shows that the distances between the environments are not significant. The PCA cluster
analysis, of which one example is shown (P1 vs. P2), shows that one environment,
PA_V_R_5, which was an high temperature iron containing sediment, does not appear to
cluster with the others. It is unclear which environmental factors lead to this uniqueness.
Other factors that were expected to lead to clustering of the environments, such as
temperature, pH, and sediment type did not appear to explain much of the variation.
Phylogenetic analysis of the representative 16s clones provided a particularly eyeopening look into the extremophile diversity of these vent sediments, especially the Bacteria.
While the bulk of the clones were related to cultured and uncultured Bacteria found
20
previously in hot springs and volcanic soils, others were related to microbes found in a wide
variety of extreme habitats: mine leaching pods, mine shafts, and compost.
The previous study on Hawaiian fumaroles collected only from caves that did not
receive any sunlight, and did not find any Cyanobacteria, and potential photosynthetic
organisms were limited to Chloroflexi, of which there were few (1). In this study, many of
the steam vents sampled in this study were open to the sky, and had access to sunlight. In
some cases the vents were simple cracks in the lava through which the steam flowed. The
presence of sunlight undoubtedly made a difference in the structure of the microbial
communities living in these vents.
This study identified a unique microbial assemblage inhabiting Hawaiian steam vents.
In the future, the chemical analysis of the vents will be performed and may elucidate some of
our findings. The microbes inhabiting the vents were similar to microbes from widely
dissimilar environments, suggesting that Hawaiian fumaroles are a microbial diversity “hot
spot”.
21
ACKNOWLEDGEMENTS
I would like to thank my wonderful committee for all their support. I would
especially like to thank Dr. Rick Bizzoco for introducing me to Hawaii and teaching me how
to sample and culture extremophile. I would like to thank Dr. Scott Kelley for his guidance
and patience throughout this project. Thanks also goes to Aaron Pietruszka for his invaluable
assistance in helping us determine sampling locations and sharing his knowledge of Kilauea
with us. Special thanks goes to the staff of Hawaii Volcanoes National Park for allowing us
access to their geothermal areas.
The Segall lab deserves special thanks for gifting me with small amounts of
restriction enzymes to test, as well as the Rowher lab for their general assistance and help.
Also I would like to thank Dr. Ralph Fueur for the use of his microscope and the Fueur lab
for their help and assistance. I could never have completed this project without the support of
the Kelley lab; many thanks for listening me talk about my project and giving me valuable
feedback and ideas. Also, Courtney Benson taught me the cloning procedure that I used and I
doubt my project would have gone so smoothly if it weren’t for her help. Finally I would like
to thank my father, John Wall, for his continued support of my educational endeavors.
22
REFERENCES
1. Benson, C. A., R. W. Bizzoco, D. A. Lipson, and S. T. Kelley. 2011. Microbial
diversity in nonsulfur, sulfur and iron geothermal steam vents. FEMS Microbiology
Ecology.76: 74–88.
2. Cole, J. R. 2009. The ribosomal database project: Improved alignments and new tools for
rRNA analysis. Nucl. Acids Res. 3: D141-D145.
3. Costello, E. K., S. R. P. Halloy, S. C. Reed, P. Sowell, and S. K. Schmidt. 2009.
Fumarole-supported islands of biodiversity within a hyperarid, high-elevation landscape
on Socompa volcano, Puna de Atacama, Andes. Appl. Envir. Microbiol. 75: 735 - 747.
4. Delmelle, P. and J. Stix. 2000. Volcanic gases. In H. Sigurdsson (ed.), Encyclopedia of
Volcanoes. Academic Press, San Diego, CA.
5. DeLong, E. F. 1992. Archaea in coastal marine environments. PNAS. 89(12): 56855689.
6. Ellis, D. G., R. W. Bizzoco, and S. T. Kelley. 2008. Halophilic Archaea determined
from geothermal steam vent aerosols. Environmental Microbiology. 10: 1582–1590.
7. Herrera, A., and C. S. Cockell. 2007. Exploring microbial diversity in volcanic
environments: A review of methods in DNA extraction. Journal of Microbiological
Methods. 70(1): 1-12.
8. Jeanthon, C., S. L. Haridon, V. Cueff, A. Banta, A. Reysenbach, and D. Prieur.
2002. Thermodesulfobacterium hydrogeniphilum sp. nov., a thermophilic,
chemolithoautotrophic, sulfate-reducing bacterium isolated from a deep-sea hydrothermal
vent at Guaymas Basin, and emendation of the genus Thermodesulfobacterium. Int. J.
Syst. Evol. Microbiol. 52: 765-772.
9. Konhauser, K. O., et al. 2002. Microbial mediation of authigenic clays during
hydrothermal alteration of basaltic tephra, Kilauea volcano. Geochem. Geophys. Geosyst.
3(12): 1075.
10. Lozupone, C. and R. Knight. 2005. UniFrac: a new phylogenetic method for comparing
microbial communities. Appl Environ Microbiol. 71(12): 8228-8235
11. Mayhew, L., D. Geist, S. Childers, and J. Pierson. 2007. Microbial community
comparisons as a function of the physical and geochemical conditions of Galápagos
Island fumaroles. Geomicrobiology Journal. 24(7): pp. 615-625
12. Miller, M. A., W. Pfeiffer, and T. Schwartz. 2010. Creating the CIPRES science
gateway for inference of large phylogenetic trees. In Proceedings of the Gateway
Computing Environments Workshop (GCE), New Orleans, LA.
13. Norris, P. R., D. A. Clark, J .P. Owen, and S. Waterhouse. 1996. Characteristics of
sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphideoxidizing bacteria. Microbiology. 142: 775-783.
23
14. Portillo, M. 2008. Microbial communities and immigration in volcanic environments of
Canary Islands. Naturwissenschaften. 95(4): 307-315.
15. Rawling, D. E. 2005. Characteristics and adaptability of iron- and sulfur-oxidizing
microorganisms used for the recovery of metals from minerals and their concentrates.
Microb Cell Fact. 4(13): 1475-2859
16. Spring, S., P. Kampfer, K. H. Schleifer. 2001. Limnobacter thiooxidans gen. nov., sp.
nov., a novel thiosulfate-oxidizing bacterium isolated from freshwater lake sediment. Int.
J. Syst. Evol. Microbiol. 51: 1463-1470.
17. Stahl, D. A. 1991. Development and application of nucleic acid probes in bacterial
systematic. In E. Stackebrandt and M. Goodfellow (ed.), Nucleic Acid Techniques in
Bacterial Systematics. John Wiley and Sons Ltd., Chichester, UK.
18. Stamatakis, A., P. Hoover, and J. Rougemont. 2008. A rapid bootstrap algorithm for
the RAxML web-servers. Systematic Biology. 75(5): 758-771.
19. Takada-Hoshino, Y., P. Schiffman, and Q. J. Fisher. 2004. An improved DNA
extraction method using skim milk from Soils that strongly adsorb DNA. Microbes.
Environ. 19: 13-19.
20. Wilson, K. H., R. B. Blitchington, and R. C. Greene. 1990. Amplification of bacterial
16S ribosomal DNA with polymerase chain reaction. J. Clin. Microbiol. 28: 1942-1946.