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Introduction

Inside cave. Copyright Michael N. Spilde

In the far corners of the lush Mayan rainforest, from the deep dark spiritual world of underground caverns, there emanates a pervasive and overpowering stench of rotten eggs. This invasive odor marks one’s proximity to the sulfurous spring cave, Cueva de Villa Luz. Every Spring, this cave is visited by hundreds of Mayan harvesters (men, women and children) who enter the sacred domain to fish thousands of pounds of sardines that will sustain them until the rains bring new crops.(18) While the Mayans reap the bounty of the caves entranceway, they dare not go beyond where the poisonous atmosphere could prove deadly. Deeper within the cave, its ceilings and walls drip sulfuric acid, its floors lie buried deep in mud and slime and at its farthest reaches, in a section deemed “Snot Heaven”, there exist vast colonies of microorganisms which dangle from the ceiling in stalactite-like formations.(16)

Description of Niche

Where located?

Cueva de Villa Luz, also known by the locals as “Cueva de la Sardina”, is a cavern in Tabasco, Mexico, near the small town of Tapijulapa. It is located within the regional park of Kolem Jaa. Somewhat isolated, the cave may be visited via buses and cabs leaving the state’s capitol of Villahermosa, which lies approximately forty miles from the town of Tapijulapa. The cave itself is located at the end of a mile-and-a-half trail from the Alamandro River, at the edge of the Chiapas highlands.(18)

Physical Conditions?

Up until microbial life was discovered below the Earth’s surface, people could not imagine or believe that life can be sustained without light energy harnessed from the sun. However, not only do cave dwelling microbes exist, but they also thrive in rocky shelters or natural openings onto or under the earth. Besides on the cave itself, these microbes live in air currents and water, as well as on animals, especially bats and insects. (1)

By exploring the great limestone cave, Cueva de Villa Luz, microbes were determined to be able to be sustained anywhere where there is water or moisture in a cave, such as flowing bodies of water, floors and surfaces of streams, deposit sites, and along water banks. Surfaces of running water leading into caves are a common place to find algae, whereas protozoa are likely to thrive in sediments. Fungal spores are abundant along the length of river banks. Ideal places for microbial growths are crossing points of air, cave surfaces, and water. Bacteria are ubiquitous in soil, sediments, and moisture films of limestone formations and of calcite speleothems. Chemoheterotrophs (such as fungi), organisms that rely on organic carbon, can be found in guano deposits, rotting wood, and water. (1)

Chemical reactions

Microbes thrive in moist areas within a cave. Chemoheterotrophs benefit from the carbon-rich environment of streaming water, deposit sites, and organic matters, such as decomposing plants. Rainwater that has soaked through into the caves provides oxygen to the hydrogen sulfide, turning the hydrogen sulfide into sulfuric acid. The hydrogen sulfide gives off a distinctive odor; one that is similar to the smell of rotten eggs. The sulfuric acids in turns disintegrate the neighboring rocks and cave walls. This phenomenon helps enlarge the cave internally and aid in the formation of limestone. In limestone creation, the rainwater mixes with carbon dioxide, forming carbonic acid. This particularly weak acid wears away the walls of limestone caves. (1,2)

Acidity and pH

Snottites. Copyright Michael N. Spilde

Water can also diffuse into sulfidic caves from the ground, bringing in hydrogen sulfide for microbes of biofilms to harvest the sulfur as energy. In the process of harnessing that energy, sulfuric acid is produced, creating a highly acidic environment. Certain biofilms prefer extremely acidic conditions like the snottites microbes, which live in exceptionally acidic environments with a pH of zero to one. For Cueva de Villa Luz specifically, the walls have a pH of 0.0 to 0.3. The snottites’ name derived from its physical attribute – resembling snots. Bacteria that reside in red clay-like goo have a less acidic pH if 2.5 to 3.9. The outer layers of biofilms are comprised of bacteria that survive by turning oxygen in the air to hydrogen sulfide. However, oxygen is detrimental to certain microbes, causing those microbes to withdraw to bottom layers. The microbes residing in the middle and lower layers consume hydrogen sulfide and releases sulfuric acid. This sulfur cycle creates a living environment that is rich in reduced sulfur. (2,4)

Temperature

Temperatures in Cueva de Villa Luz, like most caves, do not show a wide range of change. The cave is slightly warmer in the winter with an average temperature of 30 degrees Celsius and an average temperature of 28 degrees Celsius in the spring. The average ground temperature is a few (four or five) degrees Celsius warmer than the air temperature because the ground has the ability to store more heat than the atmosphere. Water sources above ground in the cave are much colder at approximately three degrees Celsius. The average cave temperature is about seventeen degrees Celsius, which is lower compared to Cueva de Villa Luz. (6,7)

Pressure, air current rate, humidity, and air pressure

Pressure in caves is dependant on temperature. Various ways that heat can enter are through the ceilings, grounds, and openings. The average air/wind flow rate is 1.90 m/s to 2.00 m/s. The average humidity is high at about 77, and the average air pressure is about 97 kPa to 100 kPa. Rocks within the cave itself serve as mediums for heat storage, while streams and pools cool down the cave, changing the pressure at those specific areas. (7)

Light source

In complete darkness, green slime glazes rock surfaces, meaning that there are microbial lives thriving in flowing water near those rocks. Although the cave itself is mostly devoid of light, there are areas pierced with the sun’s rays. These skylights are created through tiny openings to the cave and through cracks in the ceiling and walls. The sunlight provides a mean for photosynthesis, thus, leading to organic products. (4)

Influence by Adjacent Communities (if any)

Caves are not usually connected, so microbes evolve independently among caves and do not communicate with those in other caves. Within a cave, however, microbes can be genetically linked and communicate with each other. Similarity in microbial genome can be found in microbes of the same cave or other caves with comparable chemical conditions and geological make-up. (5)

Since cave microbes such as fungi and bacteria live in seclusion from the outside world and in undernourished environments, certain colonies need to mark their region. Some microbes even develop a way to produce and release toxic chemicals to protect their territory from adjacent communities. However, the microbes can associate with nearby spider webs and fungus gnats to form white filaments in the stream and microbial curtains hanging from gypsums. (1,4)

One major way that cave-dwelling microbes can be affected is by human contact. Caves that serve as tourist attractions are especially exposed to changes, which can be harmful. Humans can affect cave microbes by bringing in organic materials that can disrupt the living conditions of the microbial communities. Some examples of such organic matter include human waste (urine and feces), human cells (hair and skin), clothing fibers, and food. In contrast to synthetic fibers, cotton fibers from clothes are promptly devoured by microbes. These outside resources cause the greatest damage to the microbial colonies that can only survive in nutrient-poor conditions. The fungi that need organic input already have guano deposits from the bats and other animals living in the caves. If enough organic compounds are added, these microbes may die out. Another way humans can infringe on the native cave microbes is to introduce outside microbes into the cave and cave water sources via shoes, dirt/mud from shoes, clothes, and equipment. In contrast to synthetic fibers, cotton fibers are promptly devoured by microbes. Therefore, non-native microbes, also called transient microbes, can out-compete the native microbes if more nutrients are added to its environment. Human visits should be limited to allow outside microbes to die out along with their food source. (1)

Conditions under which the environment changes

Cave conditions remain pretty constant in darkness except near the cave openings because of variables such as light and air current. Temperature in caves like Cueva de Villa Luz is stable because of the natural protection that the cave offers from the harsh, changing weather and from the outside world. Since pressure is dependant upon temperature and cave temperature is constant, cave pressure is constant as well. Even so, most bacteria are dormant until sufficient nutrients are available. Outside factors such as human or other animal interactions and introduction of foreign microbes can greatly change and disturb native cave microbes by bringing in competition and the organic compounds that human visitors carry in changes the nutrient level of caves. Elevated nutrient levels can lead to death of microbes that can only survive in poor environments. (1)

The physical size of caves can increase over time, although very slowly. Carbonic acid erodes approximately one third of an inch of cave walls each thousand years, whereas sulfuric acid wears away cave walls about two inches every thousand years. (2)

Who Lives There?

Cave microorganisms

True caves are devoid of all light sources and therefore lack the most common source of energy supplied through photosynthesis. Cave microorganisms are consequently dependent upon alternative sources of energy derived from the surrounding atmosphere, minerals and rocks. Cave microorganisms are divided into two groups, heterotrophs and autotrophs. Although each group requires carbon compounds as a nutrient source, only the autotrophs have the ability to create the organic substances necessary for life directly from inorganic materials. While surface autotrophs usually gain their energy from the sun via photosynthesis, within the dark extremes of caves, certain autotrophic bacteria called chemoautotrophs have the ability to derive all of their energy needs from certain cave minerals.(19)

Microbial life can be observed within all types of caves. Cueva de Villa Luz contains an extraordinarily diverse population of microbes. Along its walls of limestone can be found a dense layer of microbial mucus as great as half an inch thick.(13) Sulfate reducing bacteria are present in very high numbers (105-106+) within the pores of rocks, which can run miles deep.(4)These sulfur-eating bacteria form slender white mucus-like colonies on the cave ceilings and walls. These microbial veils have been nicknamed by researchers as “snot-tites”.(15) Additionally, other sticky clusters of microbes, also facetiously named “phlegm balls” can be observed floating in subterranean streams of the cave.(11)

Other forms of bacteria such as coliform, which are abundant in animal feces and aquatic environments, survive within the main stream passage of Cueva Villa Luz, but are no longer present in the springs that flow into the cave.(4) Many of the unique and bizarre microbes encountered within the extreme environment of the cave are newly discovered and yet-to-be-identified microbes.(13)

Actinomycetes. Copyright Michael N. Spilde

In most limestone caves, a very common microbe inhabitant is the filamentous, fungal-like bacteria actinomycetes. They exist in abundance on cave walls and rocks. These microbes cluster together and give surfaces a silver-white coating.(4)

Other cave organisms

Cave animals comprise three main categories based on the amount of time they spend within the cave. Organisms that comprise the first group are called trogloxenes. These animals move freely in and out of the cave as temporary guests. Examples include bats, bears, skunks, moths and humans. The second group of cave inhabitants are called troglophiles. Troglophiles live their entire life cycle within a cave, but can also reside outside of the cave. Examples include cockroaches, beetles and millipedes. The final group, the troglobites, are considered the true cave dwellers, spending their entire lives within the dark zones of the cave. Examples include fish, shrimp, crayfish, salamanders, worms, snails, insects, bacteria, fungi and algae.(14)

In Cueva de Villa Luz, scientists have observed at least five kinds of bats, including three leaf-nosed species, vampire bats, and Mexican free-tailed bats that flutter overhead near the entry and in fresh air pockets.(18) The most widely found organisms are the midges, or tiny gnats, and the small fish. Predatory invertebrates such as spiders, fungus gnat larvae and amblypygids are also abundantly found throughout the cave.(4)

Interactions amongst microorganisms and other cave life

The snot-tite biofilms that are present throughout the caverns of Cueva de Villa Luz are comprised of thin layers of distinct microbe species. This mode of growth provides the many species of bacteria found throughout the different layers of the colony with protection. Such protection allows certain bacteria that would otherwise perish, to flourish inspite of the surrounding hostile environment of the cave.(2,11)

An added factor that makes Cueva de Villa Luz truly remarkable, is the abundance of life that flourishes within its dark interior. A mutualistic network amongst the caves inhabitants is established in which all life forms work together. Similar to an assembly line, one organism brings in the energy while another organism brings in the nutrients which then allows a third organism to supply the basic elements for still another organism to grow.(20) In Cueva de Villa Luz, the mucus-like microbial communities that are so common to this cave, form the primary link in its dense food chain. These sulfur-eating bacteria use hydrogen sulfide to create the nutrients that sustain the cave’s extensive food web. Through the process of chemosynthesis these bacteria oxidize sulfur for energy much in the same way that surface plants perform photosynthesis. These bacteria have the ability to utilize carbon dioxide, water and sulfur to support life within Cueva de Villa Luz. Small invertebrates such as the midges feed on the microbes, who in turn provide food for spiders and small fish. Water bugs along with the occasional nocturnal rodents prey on the fish. Humans, the caves largest predators, complete the food web.(18)

Interactions of cave microorganisms with the environment

Although most limestone caves are the natural result of slowly moving weakly acidified water, more recent developments reveal that some of the world’s largest caves may have been formed by bacterial species living within the caves. (13) The “snot-tites” of Cueva Villa Luz may be living proof of this recently formulated theory. These bacteria that extract all their energy from inorganic chemical reactions, combine the oxygen in the cave’s atmosphere with the hydrogen sulfide from its streams to produce sulfuric acid, the same acid found in car batteries.(15) The sulfuric acid produced by these microbes converts the limestone of the cave floors and walls into highly soluble gypsum (calcium sulfate mineral), which then breaks off into the stream, resulting in the expansion of the cave.(16)

There are a number of features that can be observed within a cave that may serve as evidence of microbial activity. Under favorable conditions, microorganisms can form large colonies visible to the eye that appear as dots on the host rock. These colonies are made up of millions of bacteria and are most abundant in moist areas. Unusual coloration on the host bedrock due to change in the surface chemistry is another common indication of microbial activity within a cave. Still another clue of the presence of microbial life is soft, powdery corrosion residues due to microbial interactions with cavern minerals. Finally, one of the most obvious indications of microbial activity is the formation of biofilms comprised of multiple layers of microbial communities held together by protective gel-like polymers. These microbial communities form complex structures that may resemble floating dumplings, slippery coatings, hair-like extensions and, as in the case of Cueva de Villa Luz, wads of snot-like goo.(3)

Microbial metabolism that is useful to the environment

Our food chain is based upon the production of food by photosynthesis. Through this process, plants are able to convert sunlight into energy, and along with carbon dioxide and water they produce the sugars that most life on Earth depends on. Bacteria such as that found in the deep recesses of caves where sunlight is non-existent use a similar method, called chemosynthesis, to create food. However, their energy is supplied by the energy produced from the breakdown of chemicals. The breaking of bonds releases energy just as the forming of bonds requires energy. The chemical reaction between hydrogen sulfide, water and oxygen results in the release of heat energy as molecular bonds are broken. Bacteria that reside within Cueva de Villa Luz use this energy to produce sugars through the combination of carbon dioxide and water. (16)

Conclusion

Cueva de Villa Luz offers a rare glimpse into uncharted, mysterious microbial kingdoms. The cave’s discovery lends living proof of the presence of minute life forms that delve deep within the Earth’s crust where the possibility of life seems impossible. Life within Cueva de Villa Luz serves as a unique and fascinating example of an ecosystem sustained primarily by inorganic reactions. Never before have biologists encountered a cave like Cueva de Villa Luz with its vast array of diverse life forms. It’s as if life itself was pitted against insurmountable survival conditions and met the challenge with miraculous proliferation.(13) The dark, stagnant, toxic ambience of the cave which obliges cave explorers to wear masks and protective clothing in order to shield themselves from the burning acidic residue and noxious fumes, has become a haven for the extremophiles that eke out an existence within its demanding confines. Within the depths of Cueva de Villa Luz’s labyrinthine caverns, may rest many of the secrets behind the origins of life on our planet, along with potential answers to puzzling questions of life throughout the universe. Although investigations into the true nature of these newly discovered microbes may take years to accomplish, their possible applications may be incredibly useful to humanity and the planet.(21)

Current Research

1. In the Science Daily article, “Snottites, Other Biofilms Hasten Cave Formation,” researchers investigate cave biofilms in the hopes of finding useful similarities between other existing biofilms such as the plaque on our teeth and the biofilms that corrode the hulls of steel ships. Researchers are especially drawn to the study of cave biofilms because, unlike those found in complex environments such as soil, cave biofilms are relatively simple. Cave biofilms are comprised of perhaps 10-20 species as opposed to complex biofilms which may contain thousands of species. This simplicity in their makeup makes them easier to study and work with. Professor Greg K. Druschel, used microelectrode voltammetry to distinguish between the multiple biofilm layers and their acidic levels. Within cave biofilms, the outer and innermost layers of the biofilm microbes produce sulfuric acid from oxygen and hydrogen sulfide while the middle layer has found a comfortable niche within the greater colony that protects these oxygen sensitive microbes from perishing. According to the experimental findings, the levels of hydrogen sulfide and sulfuric acid vary in terms of the different layers. Understanding the dissolution of calcium carbonate by sulfuric acid-producing biofilms within caves may have some bearing on how biofilms on teeth and the steel hulls of ships dissolve calcium phosphate.(2)

2. WKU Researcher Honored By National Cave Group: In 2001, Rick Fowler, a lab coordinator for Western Kentucky Biotechnology Center, presented groundbreaking results related to DNA studies at the WKU Biotechnology Center. This new research may open doors into the study of cave microorganisms all over the world. The WKU is working on developing new techniques that may aid in the identification of various cave microorganisms. These techniques involve the analysis of DNA signatures sampled from various cave environments. Cave bacteria are believed to play an important role in cave formation and cave food webs. They also have the ability to effectively remove contaminants from groundwater and thus may lead to a solution for the purification of our drinking supplies. Despite the incredible roles that cave microbes play not only within their own ecosystem but in ours as well, little is known regarding their way of living. This new research may provide scientists with a fast and relatively simple test for studying these incredibly diverse microbes.(12)

3. According to the article, “Looking inside earth for life on Mars,” by Steve Nadis, cave micro-organisms are easier to access verses their geologically comparable counterparts in deep-sea vents or Mars. The snottites biofilms are suspected of being able to flourish beneath Mars’s rocky surface that contains water resources to sustain this type of microbes. Because of the discovery of the microbial-rich community below Earth’s surface, scientists now believe that looking into Earth’s caves can lead to greater knowledge of life on present-day and pre-historic Mars. Christopher McKay, a NASA researcher, explains that approximately three to four billions years ago, Mars had a substantial amount of carbon dioxide in its atmosphere. The thick layer of carbon dioxide created a greenhouse effect on the red planet, similar to the greenhouse phenomenon on Earth, which warmed its surface and allowed water to exist. Over time, the carbon dioxide reacted with the water, resulting in carbonic acid. This acid in turned reacted with Mars’s rocky surface and formed limestone and dolomite. This carbonate formation exhausted Mars’s carbon dioxide supply, freezing the planet’s surface. Since life cannot flourish above ground anymore, scientists believe that the microbial lives on Mars shifted below ground, as evident in microbes on Earth that can only survive in their protective cave environment. (9,10)

4. Based on an EMBO reports written in 2006 published by the European Molecular Biology Organization, fungal growth on artworks of cave walls around the world is a cause for concern: destruction of human history. Paintings in a cave in Montignac, France, were in exceptional condition until it was subjected to tourism. The human interaction combined with an increase in humidity and temperature led to an infestation of Fusarium fungus and many other molds. Various treatments such as fungicide, disinfectants, and antibiotics failed to remove the dark spots created by the fungi and molds. Léauté Beasley, the founder and head of the US-based International Committee for the Preservation of Lascaux (ICPL), pointed out that modern science should look into the problem and not just art restorers. In another part of the world, the great Mayan pyramids and buildings, along with the artworks on them are under attack by biofilms. Microflora and other microbes are responsible for breaking down the Mayan limestone monuments with various metabolites created by the biofilms. The acidic products discolored and devalued the structures by dissipating the essential minerals like calcium from the limestone. Fortunately, recent studies show that even though microorganisms are the main cause of the destruction, they are the key that will lead to a resolution as well. Many researchers found out that Desulfovibrio desulfuricans and D. vulgaris, which are bacteria that can reduce sulphate in an anaerobic environment, are capable of eradicating the dark sulphate coating that can form on buildings and structures. A separate research discovered that oxalic acid creates an outer layer of calcium oxalate patina on rocks to help preserve stone structures. (8)

References

1) Microbes in Caves. Apr. 1997. Biology Department at the University of New Mexico. <http://www.caves.org/committee/conservation/www/a_biospeleogy/articles_bio/microbes.htm>

2) Penn State. "Snottites, Other Biofilms Hasten Cave Formation." ScienceDaily 13 December 2006. 28 Aug. 2008 <http://www.sciencedaily.com /releases/2006/12/061212091813.htm>

3) Barton, Hazel. "Introduction to Cave Microbiology: a Review for the Non-Specialist." Journal of Caves and Karst Studies 68.2 (2006): 43-54.

4) Investigations into the biology of Cueva de Villa Luz,near Tapijulapa, Tabasco, Mexico. . 4 August 1998. Biological Team. <http://www.i-pi.com/~diana/slime/villaluz>

5) Windows into Darkness: LSU Research in the Subsurface. 2007. Department of Geology & Geophysics. <http://www.geol.lsu.edu/Engel-geomicrobio.html>

6) Cave Climate . Riverina Environmental Education Centre. <http://www.reec.nsw.edu.au/geo/cave/caves/textcave/3acavecl.htm>

7) Hose, Louise and James A. Pisarowicz. "Cueva de Villa Luz, Tabasco, Mexico: Reconnaissance Study of an Active Sulfur Spring Cave and Ecosystem." Journal of Caves and Karst Studies 61.1 (1999): 13-21.

8) Rinaldi, Andrea. "Saving a Fragile Legacy: Biotechnology and Microbiology are Increasingly Used to Preserve and Restore the World's Cultural Heritage." PubMed Central Journals 7.11 (2006): 1075–1079.

9) Nadis, Steve. "Looking inside earth for life on Mars. " MIT's Technology Review. 100.n8 (Nov-Dec 1997): 14.3.

10) Espelie, Erin M. "What life looks like on Mars?. ." Natural History. 112.8 (Oct 2003): 6.4.

11) Snottites, phlegm balls, biofilm . Slim, Lynne H. <http://www.dentaleconomics.com/display_article/244278/56/none/none/Colum/Snottites,-phlegm-balls,-biofilm?host=www.dentalofficemag.com>

12) WKU Researcher Honored by National Cave Group . National Caves Association. <http://www.wku.edu/news/releases01/august/cave.html>

13) The walls are alive, Deep in a cave, scientists glimpse a strange new biology . Petit Charles. <http://www.well.com/user/peter/9CAVE.HTM>

14) The Biology of Caves . National Park Service. <http://www.nps.gov/ozar/forteachers/cave-biology.htm>

15) Eliot, John L. "Deadly Haven. " National Geographic. 199.5 (May 2001): 70. General Reference Center Gold. Gale. Chula Vista Public Library. 27 Aug. 2008

16) Miller, Jeanne. "Alive and Well in Mexico: The Living Cave of Villa Luz. " Odyssey. 10.5 (May 2001): 22. General Reference Center Gold. Gale. Chula Vista Public Library. 27 Aug. 2008

17) "Career Caver: An Interview with Louise Hose.(geologist)(Brief Article)(Interview). ." Odyssey. 10.5 (May 2001): 24. General Reference Center Gold. Gale. Chula Vista Public Library. 27 Aug. 2008

18) Hose, Louise D. "Cave of the Sulfur Eaters. " Natural History. 108.3 (April 1999): 54(1). General Reference Center Gold. Gale. Chula Vista Public Library. 27 Aug. 2008

19) Moor, George W. and Sullivan, Nicholas. Speleology: Caves and the Cave Environment . St. Louis: Cave Books,1997.

20) Caves Life Beneath the Forest . Barton, Hazel. Northern Kentucky University. <http://www.cavebiota.com/caveslifebeneaththeforesttranscript.htm>