O

MARIAN A.PULIDO-DE LEON, PhD
Microbial Culture Collection Museum of Natural History University of the Philippines Los Baños Project Leader University Researcher II and Curator

MANUEL M. BALDOVINO
Research Assistant Museum Technician I

CAVES
Temporary shelter Sacred place Spaces for celebration rituals of passage Burial enclosures Source of minerals CAVE Source of water Art galleries Treasure hunting Historical landmark Ecopark

of

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

CAVE 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.

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 STALAGMITES

FLOWSTONE

Types of Cave
SOLUTIONAL CAVE
Formed in rock that is soluble such as limestone, chalk, dolomite, marble, salt and gypsum.
www.commons.wikimedia.org

Bulalon Cave, Phil.

Lechuguilla Cave, USA Pahingahan Cave, Phil.

SEA CAVE
Found along coasts and are formed by wave action in zones of weakness in sea cliffs.

www.cdn.wn.com

Boracay, Philippines
www.southernthailand-all.com

Types of Cave
LAVA TUBE CAVE
Formed through volcanic activity
www.traveltohawaiireviewed.co m/image_attraction www.traveltohawaiireviewed.co m/image_attraction

Thurston Lava Tube, Hawaii

Canary Island, Hawaii

ICE CAVE
Occur in ice and under glaciers and are formed by melting
www.nsidc.org

www.endeavors.unc.edu

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 activity

Microbial colonies on 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
Nullabor Caves

Country
Australia

Microbes
α, β, γ and δ-Proteobacteria and novel microorganisms Proteobacteria, Plantomycetales, Cytophagal/Flexibacter/Bacteroides , Acidobacterium, Actinobacteria and green-sulfur bacteria Knoellia sinensis and K. subterranea (Actinobacteria) ε-Proteobacteria α, β and γ-Proteobacteria, Enterobacteriaceae, Xanthomonas, Bacillus/Coliform group and Lactobacillaceae Proteobacteria, Actinobacteria, Gram-positive bacteria Bacillus, Brevibacillus, Rhizobium, Sphingomonas, Staphylococcus, Pseudomonas, and other uncultured β-Proteobacteria

Proponents/ Year
Holmes et al. 2001

Altamira Cave

Spain

Schabereiter-Gurtner et al. 2002 Cuezva et al, 2009 Groth et al. 2002

Reed Flute

Guilin, Guangxi, China Wyoming, USA Carlsbad Caverns National Park New Mexico, USA

Lower Kane Cave Lechuguilla Spider

Engel et al. 2003 Northup et al. 2003

Llonin La Garma Kartchner Caverns

Asturias, Northern Spain Arizona, USA

Schabereiter-Gurtner et al. 2004 Ikner et al. 2006

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

fruitbats

Insectivorous bats

Insectivorous 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, To isolate, purify and identify bacterial species, To conduct and monitor in situ biofilm formation, To document the initial findings on the microbial diversity of caves in the Philippines, and To deposit the pure cultures in the Microbial Culture Collection of the Museum of Natural History (MCC-MNH), University of the Philippines Los Baños

2. 3. 4. 5.

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

Polillo Islands

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 documentation and preservation assessment,

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 singlecelled 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 sulfurconsuming 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 MCCMNH

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 IDENTIFICATION

AND

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. 2. 3. 4. Colony color or pigment production Colony margin Elevation Production of odor

MORPHOLOGICAL CHARACTERISTICS

1. 2. 3. 4.

Gram-reaction Cell formation Cell shape 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 Code
1

Description of Sampling Sites Bulalon Cave (B)
Surface swab of a cave wall 50 meters from the cave entrance Surface swab of a cave wall 100 meters from the cave entrance Surface swab of a cave stalactite 150 meters from the cave entrance Water sample from a pool/stream 100 meters from the cave entrance Water sample from a pool/stream 150 meters from the cave entrance; mats were also observed Dripping water from a big stalactite 100 meters from the cave entrance Dripping water from small stalactite 150 meters from the cave entrance

Mapanghe Cave (M)
Surface swab of a cave wall 50 meters from the cave entrance Surface swab of a cave wall 100 meters from the cave entrance Surface swab of a cave stalactite 150 meters from the cave entrance Water sample from a pool/stream 100 meters from the cave entrance Water sample from a pool/stream 150 meters from the cave entrance; mats were also observed Dripping water from a big stalactite 100 meters from the cave entrance Dripping water from small stalactite 150 meters from the cave entrance

2

3 4

5

6

7

Table 2. Total plate counts of samples collected in Bulalon and Mapanghe Caves.
Sample Code

Total Plate Count ( x 105)
BULALON MAPANGHE

EMB

R2A

EMB

R2A

1 2 3 4

376.0 98.0 770.0 46.0

430.0 172.0 765.0 90.5

11.2 1580.0 1425.0 66.5

26.0 1660.0 1040.0 69.5

5
6 7

61.0
73.0 235.0

227.0
156.0 345.0

52.0
40.0 1.6

174.0
51.0 3.6

Table 3. Probable Identities of Bacterial Isolates
Isolate Code (s) B1-4, B1-5, B1-6, B1-7, B6-3, B6-4, B6-5, M1-1, M1-3, M1-4, M2-1, M22, M6-1, M6-3, M6-4 B2-1, B2-2, B2-6, B3-1, B3-3, B4-1, B4-3, B4-6, M1-1, M1-2, M1-3, M14, M1-5 B5-3, B5-4, B6-1, B7-3, B7-4, B7-5, M1-5, M1-6 M5-1, M5-3, M5-5, M6-3, M6-4, M65, M7-1, M7-3, M7-4, M7-5, M7-6 B1-5, B3-5, B3-6, B4-7, B4-2, M1-2, M2-3, M3-5, M3-6, M5-6, M6-2 No. of Isolates 15 Probable Identity Pseudomonas aeruginosa

13

Serratia marcescens

8 11 11

Bacillus subtilis Bacillus cereus Micrococcus luteus

B2-3, B2-4, B2-5, B3-2, B3-4, B4-4, B4-5, M3-2, M3-3, M5-2, M5-4
B1-1, B1-2, B1-3, B5-1, B5-2, B6-2, B7-1, B7-2, M2-4, M2-5, M7-2, M7-7

11
12

Escherichia coli
Salmonella sp.

DRIPPING AND STANDING WATER

Escherichia coli (1000x)

Enterobacter sp. (1000x)

Serratia marcescens
(1000x)

Micrococcus luteus (1000x)

Escherichia coli (1000x)

Salmonella sp. (1000x)

Pseudomonas fluorescens (1000x)

Bacillus subtilis
(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 antimicrobial activities enzymatic and

WHAT CAN WE DO FOR THE CAVE?
1. Explore - survey the caves and make maps - study the biology and geology CAVE 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

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