O

MARIAN A.PULIDO-DE LEON, PhD

Microbial Culture Collection Project Leader
Museum of Natural History University Researcher II and
University of the Philippines Curator
Los Baños MANUEL M. BALDOVINO
Research Assistant
Museum Technician I
 CAVES
Temporary shelter
Sacred place
Spaces for celebration of
rituals of passage
Burial enclosures
Source of minerals CAVE
Source of water
Art galleries
Treasure hunting
Historical landmark
Ecopark
 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
CAVE
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 Other
Purposes. 2001)
 Cave
Extremely 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
CAVE
stalactites and stalagmites
Provide transitory or permanent sanctuary for a
range of organisms
 Cave Formation
Caves 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
CAVEin the presence of
Caves may form anywhere
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.
 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)

CAVE

STALACTITES FLOWSTONE

STALAGMITES
 Types of Cave
SOLUTIONAL CAVE

Formed in rock that is soluble

such as limestone, chalk, Bulalon Cave, Phil.
dolomite, marble, salt and
gypsum.

www.commons.wikimedia.org

Lechuguilla Cave, USA

Pahingahan Cave, Phil.

SEA CAVE

Found along coasts and are

formed by wave action in www.cdn.wn.com

zones of weakness in sea Boracay, Philippines

cliffs.
www.southernthailand-all.com
 Types of Cave
LAVA TUBE CAVE
www.traveltohawaiireviewed.co
Formed through volcanic m/image_attraction

Thurston Lava
activity www.traveltohawaiireviewed.co
m/image_attraction Tube, Hawaii

Canary Island,
Hawaii

ICE CAVE

Occur in ice and under

glaciers and are formed
by melting www.endeavors.unc.edu

www.nsidc.org
Ice Cave,
Antartica
Ice Cave,
Antartica
 CAVE LIFE

TROGLOBITES
(CAVE-LIMITED) TROGLOXENES
(USE CAVES TEMPORARILY)

TROGLOPHILES
(CAN BE PRESENT IN CAVES
AND OTHER
ENVIRONMENTS)
 Cave Microbes
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

Precipitation Corrosion Residues Structural Changes

Biofilms from streams Discoloration brought by Microbial colonies on

microbial activity the surface of a rock
Photos taken from Barton, H. Journal of Cave and Karst Studies, v. 68, no. 2, p. 43–54.
Cave Microbiodiversity
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 Microbial Diversity Researches
Caves Country Microbes Proponents/
Year
Nullabor Caves Australia α, β, γ and δ-Proteobacteria and Holmes et al. 2001
novel microorganisms

Altamira Cave Spain Proteobacteria, Plantomycetales, Schabereiter-Gurtner

Cytophagal/Flexibacter/Bacteroides et al. 2002
, Acidobacterium, Actinobacteria
and green-sulfur bacteria Cuezva et al, 2009

Reed Flute Guilin, Guangxi, Knoellia sinensis and K. Groth et al. 2002
China subterranea (Actinobacteria)

Lower Kane Cave Wyoming, USA ε-Proteobacteria Engel et al. 2003

Lechuguilla Carlsbad Caverns α, β and γ-Proteobacteria, Northup et al. 2003

Spider National Park Enterobacteriaceae, Xanthomonas,
New Mexico, USA Bacillus/Coliform group and
Lactobacillaceae

Llonin Asturias, Northern Proteobacteria, Actinobacteria, Schabereiter-Gurtner

La Garma Spain Gram-positive bacteria et al. 2004

Kartchner Caverns Arizona, USA Bacillus, Brevibacillus, Rhizobium, Ikner et al. 2006
Sphingomonas, Staphylococcus,
Pseudomonas, and other
uncultured β-Proteobacteria
Altamira (“High View”) Cave

Cuezva et al., 2009. Intl. J. Speleology 38:83-92

Lechuguilla Cave

Hyphomicrobium sp.

Leptospirillum sp.
Northup et al., 2003. Env. Micro. 5:1071-1086
 PHILIPPINE CAVES

Insectivorous Insectivorous
fruitbats bats bats

swiflets Cave lizards Cave frogs

 General Objective
To conduct initial assessments of
microbial diversity of caves in Polillo
Island, a Philippine Biodiversity
Conservation Priority Area (PBCPA).
 Specific Objectives
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
 TARGET AREA

Bulalon and Mapanghe/i Caves

Municipality of Burdeos Polillo
Islands
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.
 TARGET GROUP OF MICROBES

1. Bacteria from Cave Environs

- cave walls
- stalactites and stalagmites
- water pools
- streams and passages
2. Biofilm-forming Bacteria
 BIOFILM
- communities of bacteria or other single-
celled organisms organized in “slime”
exists whenever substances contact water

- beneficial for sewage treatment plants to

remove contaminants in water

- harmful by corroding pipes, clogging

water filters, causing rejection of medical
implants, and harboring bacteria that
contaminate drinking water

- complex layered communities of sulfur-

consuming microbes which were reported
to increase the rate of cave formation
 METHODOLOGY

Survey and Collection

Isolation, Purification and Identification

Screening and evaluation for Biofilm

Formation

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

Surface Swab
Resultant suspension (10-1 dilution)
 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
IDENTIFICATION
Preparation 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
 RESULTS
Table 1. Summary of samples collected from Bulalon and Mapanghe
Caves of Burdeos, Polillo Islands, Quezon Province.
Sample Description of Sampling Sites
Code
Bulalon Cave (B) Mapanghe Cave (M)
1 Surface swab of a cave wall 50 meters from Surface swab of a cave wall 50 meters
the cave entrance from the cave entrance

2 Surface swab of a cave wall 100 meters Surface swab of a cave wall 100 meters
from the cave entrance from the cave entrance

3 Surface swab of a cave stalactite 150 Surface swab of a cave stalactite 150
meters from the cave entrance meters from the cave entrance

4 Water sample from a pool/stream 100 Water sample from a pool/stream 100
meters from the cave entrance meters from the cave entrance

5 Water sample from a pool/stream 150 Water sample from a pool/stream 150
meters from the cave entrance; mats were meters from the cave entrance; mats were
also observed also observed

6 Dripping water from a big stalactite 100 Dripping water from a big stalactite 100
meters from the cave entrance meters from the cave entrance

7 Dripping water from small stalactite 150 Dripping water from small stalactite 150
meters from the cave entrance meters from the cave entrance
Table 2. Total plate counts of samples collected in Bulalon and
Mapanghe Caves.
Sample
Total Plate Count ( x 105)
Code
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 3. Probable Identities of Bacterial Isolates

Isolate Code (s) No. of Isolates Probable Identity

B1-4, B1-5, B1-6, B1-7, B6-3, B6-4, 15 Pseudomonas aeruginosa

B6-5, M1-1, M1-3, M1-4, M2-1, M2-
2, M6-1, M6-3, M6-4
B2-1, B2-2, B2-6, B3-1, B3-3, B4-1, 13 Serratia marcescens
B4-3, B4-6, M1-1, M1-2, M1-3, M1-
4, M1-5
B5-3, B5-4, B6-1, B7-3, B7-4, B7-5, 8 Bacillus subtilis
M1-5, M1-6
M5-1, M5-3, M5-5, M6-3, M6-4, M6- 11 Bacillus cereus
5, M7-1, M7-3, M7-4, M7-5, M7-6
B1-5, B3-5, B3-6, B4-7, B4-2, M1-2, 11 Micrococcus luteus
M2-3, M3-5, M3-6, M5-6, M6-2
B2-3, B2-4, B2-5, B3-2, B3-4, B4-4, 11 Escherichia coli
B4-5, M3-2, M3-3, M5-2, M5-4
B1-1, B1-2, B1-3, B5-1, B5-2, B6-2, 12 Salmonella sp.
B7-1, B7-2, M2-4, M2-5, M7-2, M7-7
DRIPPING AND STANDING WATER

Escherichia coli (1000x) Enterobacter sp. (1000x)

Serratia marcescens
(1000x)
 Micrococcus Escherichia coli (1000x)
luteus (1000x)

Salmonella sp. (1000x) Pseudomonas Bacillus subtilis

fluorescens (1000x) (1000x)
Screening for Biofilm
Formation
 MAJOR FINDINGS

1. Diversity of Bulalon and Mapanghe/i caves

as microbial habitats

2. Biofilm formers

3. Bacteria with high enzymatic and

antimicrobial activities
 WHAT CAN WE DO FOR
THE CAVE?
1. Explore
- survey the caves and make maps
- study the biology
CAVE and geology
2. Protect and Conserve
- clean up caves
- repair broken formations
- education & information dissemination
 SIGNIFICANT CAVES

"Significant Cave" refers to a cave which contains

materials possesses features that have
archaeological, cultural, ecological, historical or
CAVE
scientific value as determined by the DENR in
coordination with the scientific community and the
academe
 OPPORTUNITIES AND
CHALLENGES
Opportunities

Exploring the Existence of an Indigenous Microbial

Community in Caves
Using Molecular Techniques to Study Microbial
Communities and Discover Novel Organisms
Studying Microbe-MineralCAVE
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

MICROBIAL DIVERSITY: IMPACT ON CAVE LIFE AND FORMATION

In all things of Nature,
there is something
marvelous.
-Aristotle