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