Dripping with Life: Investigating Bacteria in Speleothem Carbonates from Natural and Artificial Surfaces

Background

Since I was a young child, I have been fascinated with space, space exploration, and the possibility of terrestrial life beyond our planet. I stared up into the night sky, wondering if some being in some other place in the galaxy was doing the same. At the beginning of this school year, I ran across an astrobiology article about the possibility of life on Mars (Sallstedt 2014). This article suggested that calcifying bacterial microbes on Earth may have influenced the growth of cave structures known as speleothem carbonates. Recently, NASA’s Mars Reconnaissance Orbiter sent images back to Earth of a cave system and lava tubes, which here are known to trap water. This discovery presents the possibility that extremophiles might be found in subterranean structures on Mars. If I could demonstrate that there is life in the speleothems on Earth, perhaps there could be life in the cave systems on Mars.

Previous research on extremophiles in speleothems on natural surfaces inspired me to conduct a similar study in my home state of Missouri, which is known for its vast and complex cave systems. I was fortunate enough to obtain permission to collect speleothems from both natural (a private local cave) and artificial (stone bridges in the state park) surfaces.

image 3177 3183

William collects a speleothem from a stone bridge. (left) William collects a speleothem from a local cave. (right)


Speleothem carbonates are dripstone structures found in cave systems. There are more than 250 different kinds of cave mineral deposits, but the majority of speleothems are composed of calcium carbonate in the form of calcite and aragonite (Encyclopedia Britannica). In this study, two types of speleothems were investigated: soda straws and cave coral. Soda straws are hollow, elongate, generally translucent tubes of calcite that represent the earliest growth of stalactites (Bewley 2005). They are equal in diameter to the water drops conducted along their length. Cave corals are typically small short stalks with bulbous ends, usually occurring in patches (Duckeck 2013).

Most dripstones are believed to form the same way: from dripping water, hence the name. This geochemical process begins when water in the soil reacts with soil CO2 to create weakly acidic water via the reaction: H2O + CO2 → H2CO3.  The low-pH water travels through limestone rock (calcium carbonate) and runs to the cave ceiling. The water dissolves and collects the calcium through the following reaction: CaCO3 + H2CO3 → Ca2+ + 2HCO3. The water continues to run with the newly collected calcium until degassing due to low pCO2  occurs (the removal of dissolved gases or solids from liquids, especially water or aqueous solutions) Ca2+ + 2HCO3- → CaCO3 + H2O + CO2. The precipitation of CaCO3 occurs as the water runs over cave walls. Over time, the accumulation of CaCO3 forms dripstones, including speleothem carbonates.

Speleothem formation was once thought to be a process that took hundreds of years to occur. Only in the past few years has this concept come in to question, strangely enough in artificial or man-made “cave” structures such as rock subways and under rock bridges. Once thought only to be capable of growing in caves, dripstone structures have begun to catch scientists’ attention. These speleothems have sometimes been produced in less than 50 years. In a study reported in 2013, scientists conducted experiments in a granitic subway station in Stockholm and discovered calcite-forming bacteria associated with speleothem formation on the artificial surface (Magnus Ivarsson 2013). These calcifying microbes induced the precipitation of carbonates, which began to build up and add onto the speleothem.

Hence,I posed the following question: Would speleothems obtained from artificial surfaces in central Missouri be similar in types of calcifying bacteria present and number of bacteria when compared to speleothems from a natural Missouri cave?

 

Purpose

The purpose of my research was: (1) to determine if any calcifying bacteria were present in Missouri speleothem carbonates collected from both natural surfaces (a local, privately owned cave) and artificial surfaces (80-year-old stone bridges and a concrete wall in a cave pool) by plating and culturing the samples; (2) to compare the calcifying bacterial numbers as well as the diversity present in the speleothems collected from both surfaces by using a PCR and (3) creating a clone library to identify all the bacteria present, including the calcifying species.

*Note: The soda straws that were growing in the cave took thousands of years to develop, while the soda straws that grew on the stone bridges developed in less than 80 years.

 

Hypotheses

I hypothesized that:

            (1) The speleothem carbonates collected from natural and artificial surfaces would harbor calcifying bacteria.

            (2) The speleothems collected from the artificial surfaces would have greater numbers of calcifying bacterial colony-forming units and exhibit greater diversity when compared to the natural cave surface.

 

Experimental Design

To conduct this study, Dr. Westenberg, microbiology professor at Missouri University of Science and Technology in Rolla, MO, was contacted and asked to critique the study. He provided training in microbiological technique and polymerase chain reactions. The study was conducted in his lab, using a laminar flow hood that met the Biosafety Level 2 criteria.

 

Collecting the Speleothems:

Permission was granted by the Missouri Department of Natural Resources to obtain five speleothems from two 80-year-old stone bridges located in Kaiser State Park (Figures 1 and 2). Permission was also obtained to collect speleothems from a privately owned cave (Figures 3, 4, 5 and 6) in central Missouri approximately 15 miles from the stone bridges (see Appendix A). Because of the close proximity of the two collection sites, similar weather patterns should have been present. The owner of the cave allowed three soda straws, two cave corals off of the cave wall and three cave corals from a concrete pool to be collected.

The same technique was used for collecting all samples. For each speleothem sample, a chisel and hammer were used to detach the speleothem from its surface. The sample was then placed in a labeled sterile container. After each use, the chisel was sterilized. Sterile gloves were worn at all times during collection. The samples were then refrigerated until they were transported to the lab for analysis.

img_3160_3159

Figure 1 (left)  Figure 2 (right)


figure 3 4

Figure 3 (left) Figure 4 (right)


figure 5 6

Figure 5 (left) Figure 6 (right)


 

Preparing Modified B4 Media for Plating Bacteria: Initial B4 media formula was for 1,000 ml, but it was converted to a 700-ml mixture. This media is selective for calcifying bacteria.

Original Mixture New Mixture
5 g of calcium acetate 3.5 g
1 g of yeast extract .7 g
1 g of glucose .7 g
15 g of agar 10.5 g 

*Note: The typical B4 media does not contain calcium acetate; this is the modified portion. This addition to the media makes it specifically selective for growing calcifying bacteria.

The ingredients were mixed in 1,000-mL Erlenmeyer flasks. The mixture was then autoclaved at 120ºC and 15 psi for 45 minutes. The flasks were placed in a bead bath to keep the agar melted until plating. The B4 media agar was then poured into petri dishes for plating after solidifying.

image 3215 3235 3237

Autoclave (left) Bead Bath (middle) Pouring the agar into petri dishes under the laminar flow hood (right)


 

Plating the Bacteria: All parts of the procedure were conducted in a laminar flow hood. First, a gram of each sample was crushed and suspended in saline solution. The suspension was then vortexed. One hundred microliters of the suspension were taken and placed on the B4 media. Twelve to 15 sterile glass beads were placed on the agar and gently shaken to evenly distribute the suspension. The beads were then carefully removed. The samples were plated out in triplicate. The plates were then placed in an incubator at 32ºC for a four-week period because the bacteria were slow-growing. The average number of bacterial and fungal colony-forming units (CFUs) were recorded and analyzed.

 

img 3225 3240 3258

Samples were crushed (left)  After suspension in a saline solution the samples were vortexed. (middle) The samples were plated out in triplicate. (right)


 

Preparing PCR Product: To run a PCR, it was first necessary to prepare a PCR product from each of the speleothem samples. The following procedure was used. Two microliters of primers (27F and 1392R) were added to two microliters of the DNA from speleothem samples. To this mixture, six microliters of water were added. Finally, ten microliters of master mix (DNA polymerase, buffer, and nucleotides) were added. Each mixture was placed in a thermocycler at the following cycle: 94⁰ C. for 90 seconds, 56⁰ C. for 30 seconds, 72⁰ C. for 90 seconds. It cycled for 30 cycles and then was held at 72⁰ C. for five minutes. The products were then stored at 10⁰ C. for 24 hours.

 

Preparing the Electrophoresis Gel: One hundred milliliters of TAE buffer (387.6 g of Tribase, 30 g of EDTA, 124 g of glacial acetic acid) were mixed with .8% agarose gel for electrophoresis. The mixture was boiled for five minutes in microwave. The mix was then poured and cooled to a gel. A thin layer of water was placed on top of the gel for conductance. The first well contained 10 microliters of DNA-sized standard, and each subsequent cell contained 10 microliters of prepared PCR product from each speleothem sample. The gel was then run for 60 minutes. Next, the gel was analyzed using a Foto/UV 26 trans-illuminator showing bacteria-highlighted streaks of bacterial DNA. (Figure 7)

brady_11_18_14

Figure 7


 

Data Table

Microbe Growth from Speleothem Samples
  After Two-Week Incubation Period   After Four-Week Incubation Period
  Average Number   Average Number
Speleothem Samples Bacterial CFU's Fungal CFU's   Bacterial CFU's Fungal CFU'S
Stone Bridge Soda Staw          
1 (n=3) 6 6   26 6
2 (n=3) 37 16   16 45
3 (n=3) 0 1   106 0
4 (n=3) 0 0   9 0
5 (n=3) 1 1   7 2
Average 9 5   33 9
           
Cave Soda Straws          
1 (n=3) 0 0   12 0
2 (n=3) 0 2   32 2
3 (n=3) 0 1   41 1
Average 0 1   28 1
           
Concrete Coral          
1 (n=3) 1 0   18 1
2 (n=3) 0 0   0 0
3 (n=3) 1 0   2 7
Average 1 0   7 3
           
Cave Coral          
1 (n=3) 0 0   0 0
2 (n=3) 0 1   15 0
Average 0 0   8 0

 

plate analysis

Fungi was observed in speleothems. (top left) The plates demonstrated that the amount of bacteria in speleothems ranged from few to many. (top middle and right)

 Bacteria displays zone of inhibition when surrounded by fungus. (bottom left) Some plates showed no visible growth of fungus or bacteria. (bottom middle) Some plates showed a diversity of both fungus and bacteria. (bottom right)


Graphical Analysis 

graph 1

graph 2

  

Discussion of Results

When conducting this study, it was interesting to note that a special media had to be prepared for calcifying microbes. It was also unusual to have a four-week incubation period; however, these microbes, extremophiles, are very slow-growing. Initially the focus was on bacterial presence, but as time progressed, fungi also seemed to play a role.

Fungal colony-forming units were also recorded and analyzed. Although on average there were more bacterial CFUs than fungal CFUs, fungi were present on several samples. Further review of the literature suggests that fungi are also involved in the growth of some speleothems. Several of the bacteria actually had zones of inhibition when surrounded by fungus.

The limited number of samples allowed for research purposes made statistical analysis impossible; however, several trends were seen. When comparing the average bacterial CFUs, there was very little difference between the speleothems collected from artificial surfaces and natural surfaces. Calcifying bacteria were found on samples from both types of surfaces. When comparing the average bacterial and fungal CFUs from the two-week incubation to the four-week incubation, a substantially larger number of colony-forming units were found at the four-week incubation period. On average, the stone bridge soda straws had the overall greatest number of bacterial and fungal CFUs. The soda straws as a group averaged more bacterial and fungal growth when compared to the coral speleothems. Speleothems growing on artificial surfaces, on average, had higher numbers of fungal CFUs. The increased number of both calcifying bacteria and fungi in the speleothems on the artificial surfaces was not surprising considering their rate of growth when compared to speleothems on the natural surfaces. Something had to account for the increased carbonate precipitation, which this study suggests is calcifying microbial involvement.

In the PCR analysis, electrophoresis showed that bacteria were indeed growing on or in all of the speleothem carbonate samples. One additional goal of this research was to identify the calcifying bacteria using DNA analysis. Due to faulty plasmids, the PCR products could not be cloned. This part of the research is ongoing with the original speleothem samples.

 

Statistics

Because of the limited number of speleothem samples that were allowed to be removed from both artificial and natural structures, statistics were impossible to conduct.

 

Conclusions

Based on this study, the following conclusions may be drawn:

(1) The initial hypothesis, that the speleothem carbonates collected from natural and artificial surfaces would harbor bacteria, was accepted.

(2) The second hypothesis, that the speleothems collected from the artificial surfaces would have greater bacterial diversity than those from natural cave surfaces, was rejected because the results were inconclusive.

 

Future Studies

Because my research indicated that calcifying bacteria were present in speleothems from both natural and artificial surfaces, the next step would be to identify and isolate the specific calcifying bacteria to better understand these biogeochemical processes on Earth. Possible future studies involve collecting speleothem samples from different locations in the solar system and use PCR and plating techniques to find possible life. In the future, when space travel is more common, samples might be collected from extreme conditions to look for possible life. As a future astrobiologist, I perhaps will be there when that happens.

 

Acknowledgements

I would like to thank the following people for their assistance with my project:

• Dr. David Westenberg, for being my qualified scientist and giving me the proper training and technique for my project.

• The Missouri Department of Conservation, for allowing me to collect speleothem samples from stone bridges in Kaiser State Park.

• Mrs. Constance Wyrick, for being my research advisor and helping me with my project.

  

Appendix A

Observations

Collection of Speleothems from Artificial Surfaces (80-year-old stone bridges)

Location: Camp Pin Oak Road

  • Speleothems were located on concrete, calcium patches adjacent to the actual stone.
  • Sample 2 had two speleothems growing together.
  • The color of Sample 1 was a very bright white. The color of Sample 2 was a light gray.
  • Both Samples 1 and 2 had no water dripping through at the time.

Location: Park Cabin Road

  • The stone bridge was not as tall as the bridge at Camp Pin Oak, but it was much wider.
  • Almost all the speleothems were on the east side.
  • The samples were all adjacent to the stone.
  • All samples were high with the exception of Sample 4, which was three feet above the ground where the bridge began to arch.
  • The color of Sample 3 was a bright white. The color of Sample 4 was a light brown. The color of Sample 5 was a light gray.
  • Sample 4 had water dripping through it.

Collection of Speleothems from the Local Cave in Miller County

Cave Observations:

  • Has life, including bats and amphibians.
  • Available to the public, but samples were collected away from the public access points.
  • Has electric lighting.

Soda straws location: To the left of Onyx Falls Pool, 250 yards deep into the cave, first level of the cave, not susceptible to human contact.

  • Three samples were collected, all of which had water dripping through them.
  • All samples were a very light yellow.

Concrete coral location: Onyx Falls Pool, artificial surface (built in 1974)

  • Three samples were obtained, all of which were collected with water.
  • The color of all of the cave corals was a light brown.

Cave coral locations: Onyx Circle and Moonshiner’s Dam

  • Sample 5, cave coral in Onyx Circle, probably contained iron. The color was red.
  • Sample 5, cave coral in Moonshiner’s Dam. It was black and displayed crystal growth.

 

Sample Preparation

Samples 1-5 (stone bridge soda straws)

  • Sample 1: Light green when mixed with saline solution. Easy to crush.
  • Sample 2: Light green when mixed with saline solution. Easy to crush.
  • Sample 3: Light gray when mixed with saline solution. Easy to crush.
  • Sample 4: Light tan when mixed with saline solution. Easy to crush.
  • Sample 5: Off-white when mixed with saline solution. Easy to crush.

Samples 6-8 (cave soda straws)

  • Sample 6: White when mixed with saline solution. Hard to crush.
  • Sample 7: White when mixed with saline solution. Very hard to crush.
  • Sample 8: Off-white when mixed with saline solution. Hard to crush.

Samples 9 and 10 (cave coral)

  • Sample 9: Light brown when mixed with saline solution. Hard to crush.
  • Sample 10: Medium gray when mixed with saline solution. Extremely hard to crush.

Samples 11-13 (cave coral on concrete wall)

  • Sample 11: Brown and black when mixed with saline solution. Easy to crush.
  • Sample 12: Brown and black when mixed with saline solution. Easy to crush.
  • Sample 13: Brown and black when mixed with saline solution. Easy to crush.

 

Bibliography

Achal, V., et al. “Effect of calcifying bacteria on permeation properties of concrete structures.” Microbial Biotechnology (2010): 1229-1234.

Banks, Eric D., et al. “Bacterial calcium carbonates precipitation in cave environments: A function of calcium homeostasis.” Geomicrobiology Journal (2010): 444-454.

Barton, Hazel A., et al. “Microbial life in the underworld: Biogenicity in secondary mineral formations.” Geomicrobiology Journal (2001): 359-368.

Baskar, S.B. “Evidence for microbial involvement in the genesis of speleothem carbonates, Borra Caves, Visakhapatnam, India.” Current Science (2007): 350-355.

Bewley, Djuna. “Soda Straws.” The Virtual Cave, 5 May 2005. Web. 28 Apr. 2015.

Blyth, Alison J., et al. “Molecular evidence for bacterial mediation of calcite formation in cold high-altitude caves.” Geomicrobiology Journal (2008): 101-111.

Cacchio, Paola, et al. “Involvement of microorganisms in the formation of carbonate speleothems in the Cervo Cave.” Geomicrobiology Journal (2004): 497-509.

“Cave Deposit.” Encyclopædia Britannica Online. Encyclopædia Britannica Inc., 2015. Web. 28   Apr. 2015.

Duckeck, Jochen. “Cave corals.” Show Caves, 12 Sept. 2013. Web.

Groth, I., et al. “Geomicrobiological study of the Grotta dei Cervi, Porto Badisco, Italy.” Geomicrobiology Journal (2001): 241-258.

Lee, Y.N. “Calcite production by bacillus Amyloliquefaciens.” The Journal of Microbiology (2003): 361-363.

Legatzki, A., et al. “Factors influencing observed variations in the structure of bacterial communities on calcite formations in Kartchner Caverns, AZ, USA.” Geomicrobiology Journal (2012): 422-434.

Magnus Ivarsson, J.E. “Kungstradgarden, a granitic subway station in Stockholm: Its ecosystem and speleothems.” Speleological Research and Activities in Artificial Underground (2013): 217-220.

Melim, Leslie A., et al. “Evidence for microbial involvement in pool finger precipitation, Hidden Cave, New Mexico.” Geomicrobiology Journal (2001): 311-329.

Northup, Diana E., et al. “Geomicrobiology of caves: A review.” Geomicrobiology Journal (2001): 199-222.

Ortiz, M. “Profiling bacterial diversity and taxomic composition on speleothem surfaces in Kartchner Caverns, AZ.” Microbe Ecology (2013): 371-383.

Rusznyak, Anna, et al. “Calcite biomineralization by bacterial isolates from the recently   discovered pristine karstic Herrenberg cave.” Appl Environmental Biology (2012): 1-15.

Sallstedt, T., et al. “Speleothem and Biofilm Formation in a Granite/Doleration Cave, Northern Sweden.” International Journal of Speleology 43.3 (Sept. 2014): 305-313.

Wang, J.Y., et al. “Diatomaceous earth as a protective vehicle for bacteria applied for self-healing concrete.” Microbial Biotechnology (2012): 567-577.

Zamarreno, Dania V., et al. “Carbonate crystals precipitated by freshwater bacteria and their use as a limestone consolidant.” Applied and Environmental Microbiology (2009): 5981-         5989.