View
486
Download
5
Embed Size (px)
DESCRIPTION
A report on the UPLB Basic Research Program funded study to conduct initial assessments of microbial diversity of caves in Polillo Island, a Philippine Biodiversity Conservation Priority Area (PBCPA). Among the specific objectives are to: 1.) collect biofilm samples from cave environs (streams, pool of standing water, underwater passages, dripping water and rocks) of Mapanghe and Bulalon Caves in Polillo Islands, 2.) isolate, purify and identify bacterial species, 3.) conduct and monitor in situ biofilm formation, 4.) document the initial findings on the microbial diversity of caves in the Philippines, and 5.) deposit the pure cultures in the Microbial Culture Collection of the Museum of Natural History (MCC-MNH), University of the Philippines Los Baños.
Citation preview
O
Project Leader
University Researcher II and
Curator
Microbial Culture Collection
Museum of Natural History
University of the Philippines
Los Baños
Research Assistant
Museum Technician I
MARIAN A.PULIDO-DE LEON, PhD
MANUEL M. BALDOVINO
CAVE
CAVES
Temporary shelter
Sacred place
Spaces for celebration of
rituals of passage
Burial enclosures
Source of minerals
Source of water
Art galleries
Treasure hunting
Historical landmark
Ecopark
CAVE
Cave“Any naturally occurring void, cavity, recess or
system of interconnected passages beneath the
surface of the earth or within a cliff or ledge and
which is large enough to permit an individual to
enter, whether or not the entrance, located
either in private or public land, is naturally
formed or man-made. It shall include any natural
pit, sinkhole or other feature, which is an
extension of the entrance. The term also
includes cave resources therein, but not any vug,
mine tunnel, aqueduct or other man-made
excavation.”
(Section 3. Definition of Terms. Republic Act 9072. An Act to
Manage and Protect Caves and Cave Resources and for OtherPurposes. 2001)
CAVE
CaveExtremely low in nutrients
Consist of sulfur, iron and manganese deposits
Often adorned with calcium carbonate
formations produced through slow precipitation,
including the most common and well-known
stalactites and stalagmites
Provide transitory or permanent sanctuary for a
range of organisms
CAVE
Cave FormationCaves take millions of years to form
Formed by geologic processes and involves a
combination of chemical processes, erosion
from water, tectonic forces, microorganisms,
pressure and atmospheric influences
Caves may form anywhere in the presence of
soluble rock
Cave formation occurs because limestone
dissolves under the action of rainwater and
groundwater charged with H2CO3 (carbonic
acid)
Secondary mineral deposits in caves are called
speleothems.
CAVE
SPELEOTHEM“Any natural mineral formation or deposit occurring
in a cave or lava tube, including but not limited to any
stalactite, stalagmite, helictite, cave flower, flowstone,
concretion, drapery, rimstone or formation of clay or
mud.” (Republic Act 9072, 2001)
STALACTITES
STALAGMITES
FLOWSTONE
Lechuguilla Cave, USA
Pahingahan Cave, Phil.
Bulalon Cave, Phil.
Types of Cave
SOLUTIONAL CAVE
Formed in rock that is soluble
such as limestone, chalk,
dolomite, marble, salt and
gypsum.
SEA CAVE
Found along coasts and are
formed by wave action in
zones of weakness in sea
cliffs.www.southernthailand-all.com
Boracay, Philippines
www.cdn.wn.com
www.commons.wikimedia.org
Types of Cave
ICE CAVE
LAVA TUBE CAVE
Formed through volcanic
activityThurston Lava
Tube, Hawaii
Canary Island,
Hawaii
Occur in ice and under
glaciers and are formed
by melting
www.traveltohawaiireviewed.co
m/image_attraction
www.traveltohawaiireviewed.co
m/image_attraction
www.endeavors.unc.edu
Ice Cave,
Antartica
www.nsidc.org
Ice Cave,
Antartica
CAVE LIFE
TROGLOBITES (CAVE-LIMITED) TROGLOXENES
(USE CAVES TEMPORARILY)
TROGLOPHILES (CAN BE PRESENT IN CAVES
AND OTHER
ENVIRONMENTS)
Microorganisms can be TRANSIENT or those
that ride into caves on air currents, in water
flows, on insects, on bats, and through humans.
Microorganisms can be RESIDENT or those
that occur as native inhabitants of a cave and
depend solely on the resources within the cave
for survival.
Cave Microbes
Biofilms from streams Discoloration brought by
microbial activityMicrobial colonies on
the surface of a rockPhotos taken from Barton, H. Journal of Cave and Karst Studies, v. 68, no. 2, p. 43–54.
Cave Microbes
Precipitation Corrosion Residues Structural Changes
Focus on the adaptation of microbial
communities and individual microbial
species to near-starvation conditions
Examine the microbial contribution to cave
ecology, mineral formation and ecosystem
bioenergetics
Find novel microbes
Understand humans’ impact on cave
microbes
Cave Microbiodiversity
Caves Country Microbes Proponents/
Year
Nullabor Caves Australia α, β, γ and δ-Proteobacteria and
novel microorganisms
Holmes et al. 2001
Altamira Cave Spain Proteobacteria, Plantomycetales,
Cytophagal/Flexibacter/Bacteroides
, Acidobacterium, Actinobacteria
and green-sulfur bacteria
Schabereiter-Gurtner
et al. 2002
Cuezva et al, 2009
Reed Flute Guilin, Guangxi,
China
Knoellia sinensis and K.
subterranea (Actinobacteria)
Groth et al. 2002
Lower Kane Cave Wyoming, USA ε-Proteobacteria Engel et al. 2003
Lechuguilla
Spider
Carlsbad Caverns
National Park
New Mexico, USA
α, β and γ-Proteobacteria,
Enterobacteriaceae, Xanthomonas,
Bacillus/Coliform group and
Lactobacillaceae
Northup et al. 2003
Llonin
La Garma
Asturias, Northern
Spain
Proteobacteria, Actinobacteria,
Gram-positive bacteria
Schabereiter-Gurtner
et al. 2004
Kartchner Caverns Arizona, USA Bacillus, Brevibacillus, Rhizobium,
Sphingomonas, Staphylococcus,
Pseudomonas, and other
uncultured β-Proteobacteria
Ikner et al. 2006
Cave Microbial Diversity Researches
Cuezva et al., 2009. Intl. J. Speleology 38:83-92
Altamira (“High View”) Cave
Lechuguilla Cave
Hyphomicrobium sp.
Leptospirillum sp.
Northup et al., 2003. Env. Micro. 5:1071-1086
Insectivorous bats
Insectivorous bats
Cave frogs
fruitbats
Cave lizardsswiflets
PHILIPPINE CAVES
General Objective
To conduct initial assessments of
microbial diversity of caves in Polillo
Island, a Philippine Biodiversity
Conservation Priority Area (PBCPA).
1. To collect biofilm samples from cave environs (streams,
pool of standing water, underwater passages, dripping
water and rocks) of Mapanghe and Bulalon Caves in
Polillo Islands,
2. To isolate, purify and identify bacterial species,
3. To conduct and monitor in situ biofilm formation,
4. To document the initial findings on the microbial
diversity of caves in the Philippines, and
5. To deposit the pure cultures in the Microbial Culture
Collection of the Museum of Natural History (MCC-MNH),
University of the Philippines Los Baños
Specific Objectives
Polillo
Islands
TARGET AREA
Bulalon and Mapanghe/i Caves
Municipality of Burdeos
Polillo Islands
Quezon Province
- 2 out 18 caves in
Quezon and 2 out of
10 caves Burdeos
listed in 2001 Phil.
Protected Areas &
Wildlife Resources
Criteria for Selection of Target Areas
1. Polillo Islands categorized as Extremely High
critical priority level
2. Municipality of Burdeos as karstic area
3. Umbrella Research Program of the Museum of
Natural History
4. Subject of biodiversity assessment,
documentation and preservation
5. Accessibility of two caves, Mapanghe/I and Bulalon
Bulalon Cave
Polillo
Islands
Alviola et al.
Mapanghe/i Cave
Alviola et al.
1. Bacteria from Cave Environs
- cave walls
- stalactites and stalagmites
- water pools
- streams and passages
2. Biofilm-forming Bacteria
TARGET GROUP OF MICROBES
- communities of bacteria or other single-celled organisms organized in “slime”exists whenever substances contact water
- beneficial for sewage treatment plants toremove contaminants in water
- harmful by corroding pipes, cloggingwater filters, causing rejection of medicalimplants, and harboring bacteria thatcontaminate drinking water
- complex layered communities of sulfur-consuming microbes which were reportedto increase the rate of cave formation
BIOFILM
METHODOLOGY
Survey and Collection
Isolation, Purification and Identification
Screening and evaluation for BiofilmFormation
In situ Biofilm Formation
Depository of Pure Cultures to MCC-MNH
1. SURVEY AND COLLECTION
Preparation and sterilization of
materials
1. 25 cm2 template (made of cardboard)
2. 10 mL 0.1% sterile peptone water
3. sterile swabs
Laying the template over an area;
Swabbing of the area using sterile
cotton swabs and dipping swabs (1st
and 2nd) in 0.1% peptone water
Resultant suspension (10-1 dilution)
Surface Swab
Preparation and sterilization of
materials
1. Sterilized Sumilon tubes
2. Ice box with dry ice (storage of
samples)
Collection of 10-15 mL samples from
dripping water and/or water from pools
and streams
Storage of samples prior to use
Water Collection
2. ISOLATION, PURIFICATION AND
IDENTIFICATIONPreparation and sterilization of
materials and culture media
1. R2A and Eosin Methylene Blue Agar
2. 0.1% peptone water 10-1 to 106
dilutions
3. Nutrient Agar (for masterplates)
Incubation at 28 – 30 C for 18-48 hr
Total Plate Count (CFU/mL or CFU/cm2)
Purification of representative isolates
Master plate of representative colonies
Observation of Isolates:
Cultural, Morphological, Biochemical
CULTURAL CHARACTERISTICS
1. Colony color or pigment production
2. Colony margin
3. Elevation
4. Production of odor
MORPHOLOGICAL CHARACTERISTICS
1. Gram-reaction
2. Cell formation
3. Cell shape
4. Spore formation
BIOCHEMICAL CHARACTERISTICS
1. Catalase reaction
2. Fermentation of sugars
3. Other test for further identification of
microbial species
3. In-situ Biofilm Formation
Sample
Code
Description of Sampling Sites
Bulalon Cave (B) Mapanghe Cave (M)
1 Surface swab of a cave wall 50 meters from
the cave entrance
Surface swab of a cave wall 50 meters
from the cave entrance
2 Surface swab of a cave wall 100 meters
from the cave entrance
Surface swab of a cave wall 100 meters
from the cave entrance
3 Surface swab of a cave stalactite 150
meters from the cave entrance
Surface swab of a cave stalactite 150
meters from the cave entrance
4 Water sample from a pool/stream 100
meters from the cave entrance
Water sample from a pool/stream 100
meters from the cave entrance
5 Water sample from a pool/stream 150
meters from the cave entrance; mats were
also observed
Water sample from a pool/stream 150
meters from the cave entrance; mats were
also observed
6 Dripping water from a big stalactite 100
meters from the cave entrance
Dripping water from a big stalactite 100
meters from the cave entrance
7 Dripping water from small stalactite 150
meters from the cave entrance
Dripping water from small stalactite 150
meters from the cave entrance
RESULTS
Table 1. Summary of samples collected from Bulalon and Mapanghe
Caves of Burdeos, Polillo Islands, Quezon Province.
Sample
CodeTotal Plate Count ( x 105)
BULALON MAPANGHE
EMB R2A EMB R2A
1 376.0 430.0 11.2 26.0
2 98.0 172.0 1580.0 1660.0
3 770.0 765.0 1425.0 1040.0
4 46.0 90.5 66.5 69.5
5 61.0 227.0 52.0 174.0
6 73.0 156.0 40.0 51.0
7 235.0 345.0 1.6 3.6
Table 2. Total plate counts of samples collected in Bulalon and
Mapanghe Caves.
Isolate Code (s) No. of Isolates Probable Identity
B1-4, B1-5, B1-6, B1-7, B6-3, B6-4,
B6-5, M1-1, M1-3, M1-4, M2-1, M2-
2, M6-1, M6-3, M6-4
15 Pseudomonas aeruginosa
B2-1, B2-2, B2-6, B3-1, B3-3, B4-1,
B4-3, B4-6, M1-1, M1-2, M1-3, M1-
4, M1-5
13 Serratia marcescens
B5-3, B5-4, B6-1, B7-3, B7-4, B7-5,
M1-5, M1-6
8 Bacillus subtilis
M5-1, M5-3, M5-5, M6-3, M6-4, M6-
5, M7-1, M7-3, M7-4, M7-5, M7-6
11 Bacillus cereus
B1-5, B3-5, B3-6, B4-7, B4-2, M1-2,
M2-3, M3-5, M3-6, M5-6, M6-2
11 Micrococcus luteus
B2-3, B2-4, B2-5, B3-2, B3-4, B4-4,
B4-5, M3-2, M3-3, M5-2, M5-4
11 Escherichia coli
B1-1, B1-2, B1-3, B5-1, B5-2, B6-2,
B7-1, B7-2, M2-4, M2-5, M7-2, M7-7
12 Salmonella sp.
Table 3. Probable Identities of Bacterial Isolates
Enterobacter sp. (1000x)
Serratia marcescens (1000x)
Escherichia coli (1000x)
DRIPPING AND STANDING WATER
Micrococcus luteus (1000x)
Escherichia coli (1000x)
Salmonella sp. (1000x) Pseudomonas fluorescens (1000x)
Bacillus subtilis (1000x)
Screening for Biofilm
Formation
1. Diversity of Bulalon and Mapanghe/i cavesas microbial habitats
2. Biofilm formers
3. Bacteria with high enzymatic andantimicrobial activities
MAJOR FINDINGS
CAVE
WHAT CAN WE DO FOR
THE CAVE?
1. Explore
- survey the caves and make maps
- study the biology and geology
2. Protect and Conserve
- clean up caves
- repair broken formations
- education & information dissemination
CAVE
SIGNIFICANT CAVES
"Significant Cave" refers to a cave which contains
materials possesses features that have
archaeological, cultural, ecological, historical or
scientific value as determined by the DENR in
coordination with the scientific community and the
academe
MICROBIAL DIVERSITY: IMPACT ON CAVE LIFE AND FORMATION
CAVE
OPPORTUNITIES AND
CHALLENGESOpportunities
Exploring the Existence of an Indigenous Microbial
Community in Caves
Using Molecular Techniques to Study Microbial
Communities and Discover Novel Organisms
Studying Microbe-Mineral Interactions in Caves
Caves as Laboratories for Developing Life Detection
Using Studies of Microbes in Caves to Captivate Young
Learners
Challenges
Moving Beyond Who’s Home Studies
Culture-independent versus culture-dependent studies
Funding
Need for Microbial Speleologists
In all things of Nature,
there is something
marvelous.
-Aristotle