The subject of this NSF Microbial Observatory (MO) is Kartchner Caverns in Benson, Arizona, one of the top ten carbonate caves in the world in terms of mineral and cave formation diversity. Until they are discovered, underground caves are ancient, fragile, nutrient-poor, and isolated systems with little or no connection to surrounding environments. Upon discovery and without proper management, damage to these systems can be irreversible. Scientifically, caves are of interest because it is likely they harbor undiscovered and novel microorganisms and because these insulated systems are devoid of light, and may harbor unique microbial activities as well. For the general public, caves harbor fascinating and beautiful formations that generate interest in science and the preservation of unique and fragile ecosystems. The objectives of the Kartchner Caverns MO are: 1) to identify novel organisms associated with different parts of the cave including formations, walls, and mud using both culture methods and advanced molecular tools to analyze DNA signatures, 2) to construct a complete DNA (metagenomic) library and database for correlating specific DNA sequences with microbial activities in the cave environment (e.g., participation in creating the formations), 3) to examine how microbial communities change across defined carbon gradients in the cave to help define how tourism, which brings in substantial amounts of extra organic carbon (e.g., lint, hair, skin cells), has impacted the cave, and 4) to analyze soap-like molecules (biosurfactants) produced by formation-associated microorganisms and incorporate their contributions into modeling the growth process of the formations.
Fundamental understanding of the biology of cave systems, in general, is lacking. This is a major impediment to sustainable public development of these unique and delicate ecosystems. Thus, the scope of this research is both timely and comprehensive in regard to the study of cave microbial diversity. The broader impacts of this project include the establishment of both microbial and DNA (metagenomic) libraries that will comprise the most detailed, surface-specific cave collections to date. These libraries will provide insights into the selective forces that drive the microbial biology of subterranean nutrient-deprived existence. The libraries will also be made widely available for screening and data mining for biotechnological and biomedical applications. The studies along various gradients (inorganic and organic carbon) will provide insight into how environmental factors impact the associated microbial communities and the attendant health and stability of the cave system. Finally, the integration of biosurfactant production into models of formation development represents one of the first efforts to understand microbial contributions to formation growth. In summary, this MO will establish a world-wide resource for the advancement of contemporary research on cave microbiology and provide valuable tools for the global preservation of caves and cave formations. The broader impacts of this MO go far beyond research. Educational impacts include training of post-doctoral, graduate, and undergraduate students to prepare a new generation of scientists in modern microbiological methods to address contemporary challenges in cave conservation and microbial diversity in other fragile, unique environments. Outreach efforts will impact several hundred thousand people per year and include: (i) an annual Kartchner Caverns lecture series for Park personnel and docents to provide a microbiology background and an update on project progress and findings which will then be passed on to Park visitors (~200,000/year), (ii) a high-quality interactive display for the Kartchner Educational Center to provide Park visitors a unique opportunity to learn about cave microbiology, the MO, and recent findings, (iii) production of a high-definition DVD by the University of Arizona nationally acclaimed PBS affiliate, KUAT, that highlights the MO project and its role in understanding microbial diversity in Kartchner Caverns, (iv) development of a module for Arizona State Parks educational curriculum that is targeted at grades 7-12, and (v) a Kartchner Caverns MO website with reciprocal links to the Kartchner Caverns website and the Friends of Kartchner Cavern website (together, these websites receive over 50K hits monthly).
The food web on the Earth’s surface is ultimately fueled by energy from the sun which is used to reduce atmospheric carbon dioxide (CO2) to organic carbon sugars through photosynthesis, also known as photoautotrophy. In contrast, caves are dark, subterranean ecosystems – an extreme environment - constrained by the absence of sunlight and organic matter. Conventional understanding posits that the primary source of food for these subterranean microbial communities is organic carbon percolating into the cave with drip water from surface soils. Microorganisms that generate energy based on the oxidation of such organic compounds, are known as chemoheterotrophs. Research results suggest that this conventional understanding must be modified and that in fact, the microbial communities colonizing calcite speleothems (e.g. stalactites, stalagmites) and rock surfaces include autotrophic populations that fix CO2 from the cave atmosphere using sources of energy other than sunlight. These autotrophs, known as chemoautotrophs, oxidize inorganic chemicals such as ammonia or ferrous iron to generate needed energy. Diversity surveys in Kartchner Caverns show that bacteria are the most abundant microorganisms in speleothem communities followed by archaea, fungi and viruses. Only 16% of the bacteria in the soil from above the cave were detected in any of the cave communities surveyed suggesting that caves are colonized by novel microbial communities distinct from those present in surface soils. A second intriguing finding is that the archaea are significantly more abundant in the cave than in the soil above the cave. Archaea, are similar in size to bacteria but have evolved quite separately, showing adaptation in particular to extreme environments like Kartchner Caverns. Finally, many of the cave microbes were able to precipitate calcite, leading us to speculate that microbes may facilitate speleothem growth and contribute to the diversity of structures seen in caves. Four speleothems and one rock surface were selected and sampled for extensive DNA sequencing (metagenome analysis) to probe the functional metabolic potential of these novel subterranean communities, in other words to ask the question "How do these communities make a living?" Strikingly, we found genes from all six known CO2-fixation pathways. Further, the RuBisCo gene which belongs to the most widespread autotrophic pathway in bacteria and eukaryotes (the Calvin Benson Bashan pathway) was significantly more abundant in the speleothem metagenomes than in metagenomes from soil and deep ocean environments. These results suggest that cave microbial communities have diverse mechanisms for fixing CO2 and thus are not completely dependent on organic carbon supplies from the surface. We next examined the Kartchner environmental and metagenome data for clues to what inorganic components in the cave might be supplying the energy needed for CO2 fixation. Kartchner Caverns drip-water collected over a 12-month period contained extremely low levels of organic carbon, but significantly higher levels of nitrogen, permitting speculation that nitrogen may function as both a nutrient and an energy source for this cave ecosystem. Examination of nitrogen cycling genes showed those for ammonia oxidation (nitrification) were well represented in cave communities. Nitrification is the two-step microbially catalyzed conversion of ammonia to nitrate and the energy produced is used by chemoautotrophic microbes to fix CO2. Both archaeal and bacterial genes for the first step in this reaction, ammonia oxidation to nitrite, were detected in the metagenome. Analysis of these genes indicated that archaea are the dominant cave ammonia-oxidizers. The second step in the energy-producing nitrification process (nitrite to nitrate) was also present but was dominated by bacterial genes highly similar to those found in the bacterium Nitrospira defluvii. Community surveys confirmed metagenome results finding ammonia-oxidizing archaea and Nitrospira in numerous speleothem communities throughout the cave. The Kartchner Caverns Microbial Observatory has produced the first definitive proof that carbonate cave communities contain diverse autotrophic (CO2 fixation) capabilities supported by a nitrogen-based energy strategy. In addition, many of the bacteria and archaea identified in this study were specifically adapted to growth under low-substrate conditions. The intellectual merit of this work is the characterization of novel microbial communities in carbonate caves that are fueled by an alternative energy paradigm and specifically adapted to oligotrophic (starvation) conditions. The Kartchner communities contained an abundance of organisms and genes with low similarity to any previously identified organisms suggesting that these communities may harbor as yet undiscovered metabolic capabilities. The broader impact of studying this novel subterranean ecosystem is to provide an opportunity to think outside the box and expand our conception of life strategies potentially sustaining poorly understood ecosystems such as those found on other planets. Two additional outcomes include the creation of an interactive kiosk for the Arizona State Parks Discovery Center at Kartchner Caverns and a Microbial Observatory website highlighting the research discoveries. The kiosk will allow 200,000 annual cave visitors to explore cave microbiology and the techniques used by microbiologists to study new ecosystems.
