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Low-diversity microbial communities in Frasassi offer a promising model system for studying biogeochemical cycling in chemosynthetically fueled, low-oxygen, sulfidic environments. Microbial sulfur oxidation occurs in Frasassi as part of a larger sulfur cycle that, above the water table, stimulates cave formation through the production of sulfuric acid. Sulfuroxidizing microbes commonly use either oxygen or nitrate as electron acceptors. Metagenomic studies of Frasassi biofilms revealed that complete sulfur oxidation pathway and some nitrate reduction genes exist within the microbial community (Hamilton, et al 2015). There are two known dissimilatory nitrate reduction pathways: denitrification and dissimilatory nitrate reduction to ammonia (DNRA). In Frasassi, H2S (100-800 µM) and Sº (20-80% dry mass of biofilms) are available in high concentrations, whereas oxygen (< 2-60 uM) and nitrate (< 0.06 µM) concentrations are much lower. Therefore we expect to find evidence of DNRA as an active nitrate reduction pathway, following the equation:


H+ + H2O + NO3+ HS→ NH4+ +SO42-                                             ΔG°= -448 kJ/molrxn


Sulfur oxidation would drive the need for nitrate and create large amounts of energy, possibly explaining why nitrate is generally found below detection limit. Using previously binned metagenomic data from Frasassi biofilm sample PC08-64, I searched for homology with functional genes related to sulfur and nitrogen cycling. Genes necessary for DNRA are present in Bin 14, which is almost complete based on the presence of a full complement of amino acid tRNA synthetase genes. Bin 14 also contained a 16S rRNA gene that was closely related to Metallibacterium of the Xanthomonadaceae family. This genus has not been investigated at Frasassi before. Although previous FISH surveys have not detected large unidentified populations, the presence of a complete or nearly complete Metallibacterium genome bin suggests that it is abundant, a hypothesis that could be tested in future work using FISH and by further analyzing EMIRGE reconstructed 16S rRNA data.
















List of Figures                                                                                                                         v

List of Tables                                                                                                                          vi

Preface and Acknowledgements                                                                                                vii


Chapter 1. Introduction

1.1 Geologic history                                                                                                    1

1.2 Frasassi Water Table                                                                                            2

1.3 Sulfuric Acid Speleogenesis                                                                                    3  1.4 Microbes in Frasassi                                                                                      5

1.5 Figures                                                                                                                  7


Chapter 2. Dissimilatory nitrate reduction to ammonia in Frasassi microbial communities


2.1. Introduction                                                                                                            10

2.2. Methods

2.2.1 Sample collection and geochemistry                                                              14

2.2.2 DNA Extraction                                                                                                15         2.2.3 Metagenomic Sequencing                                                                  15

2.2.4 Phylogenetic and Gene Analyses                                                                   16

2.2.5. ARB and MEGA Tree Building                                                                    17

2.3. Results and Discussion

2.3.1 Redox Equations and Energy Calculations                                                    18

2.3.2. Metagenomic Analyses                                                                                    19                     2.3.3. Phylogeny of 16S rRNA genes                                                                         22

2.4. Conclusion                                                                                                              25

2.5. Figures and Tables                                                                                                 28

2.6. Redox Equations and Energy Calculations                                                            45

2.7. References                                                                                                              48


Appendix A: Fluorescence in situ hybridization probe design for Thiobacillus Baregensis-like

Frasassi species                                                                                                                          52


Appendix B: Culturing of Epsilonproteobacteria                                                                      55



1.1.      Geologic History   

The Frasassi cave system is a network of caves with more than 25 kilometers of passage located on the eastern side of the Apennine Mountains, 40 kilometers away from the Adriatic Sea in the Umbria-Marche region in central Italy (Galdenzi and Maruoka, 2003). Approximately 100 caves have been identified and mapped along the Sentino River that runs through the Frasassi Gorge, a 2 km long and 500 m deep canyon cut down the center of a small anticlinal ridge (Montanari, Galdenzi, and Rossetti, 2002) (Figure 1.1). The average rainfall is about 1000 mm/year with precipitation usually reaching a maximum in autumn and spring and a minimum in summer during which evaporation exceeds precipitation (Galdenzi and Maruoka, 2003).

The cave is mainly carved out of the 600 to 1000 meter thick Calcare Massiccio

Formation, a Jurassic era rock unit made of a homogeneous (99% pure) calcium carbonate (Galdenzi and Marouka, 2003) (Figure 1.2). The Calcare Massiccio Formation is highly permeable due to high syngenetic porosity and a well-developed network of fractures (Galdenzi, 2005). The depositional environment of this pure limestone was a carbonate platform similar to that of modern-day Bahamas (Schlager and Ginsburg, 1981). This unit sits above the Triassic Anidriti del Burano Formation, an anhydrite-rich unit that is approximately

2000 meters thick and provided the sulfide to the cave system (Galdenzi, 2005) (Figure 1.2).

After the Calcare Massiccio Formation was deposited, the region went through a period of extension caused by the rifting of the European and African plates from 100-50 Ma (Montanari, et al., 2002). The extension resulted in a network of normal faults that produced horst and grabens. In the Miocene, the area shifted from extension to compression as the European and African plates converged. Compression reactivated normal faults zones associated with prior rifting and created reverse faults, sparking the orogeny of the Apennine Mountains (Eastman, 2007; Galdenzi, 2005). The Frasassi anticline was formed due to the deformation associated with plate conversion and subsequent uplift of the sedimentary basins in the late Miocene. The caves developed mainly in the eastern limb of this anticline because the fault network concentrated water on this side (Galdenzi and Maruoka, 2003).


1.2             Frasassi          Water              Table

The cave aquifer is composed of two water types: carbonate and sulfidic. The carbonate water originates from meteoric water that filters down through the limestone and is oxygenated. On the other hand, the sulfidic groundwater that characterizes the main aquifer originates from the Calcare Massicico and upper Maiolica Formations at the core of the anticline. It is higher in salinity (up to 2 g/L) and contains sulfate (up to 2.5 mM/L) and hydrogen sulfide (up to 0.5 mM/L) (Galdenzi and Maruoka, 2003). These dissolved components are mainly acquired as groundwater filters through the underlying Anidriti del Burano Formation, which is composed of anhydrite (Galdenzi and Marouka, 2003) (Figure 1.3). Deterium (δD), oxygen (δ18O), and tritium isotope signatures of water suggest that the sulfidic groundwater originates from meteoric water in a recharge area located at higher altitudes with a short residence time in the aquifer (Tazioli, et al., 1990).

The groundwater flow composed of these two waters is generally very slow with turbulent streams found in the eastern part of the cave level with the river (Galdenzi, 2003).

Water levels, conductivity and temperature of the sulfidic streams are correlated with precipitation, causing the meteoric water seepage water to vary for 30-60% throughout the year (Sarbu, et al. 1994; Galdenzi and Marouka, 2003; Galdenzi, 2005). Because the sulfidic groundwater is higher in salinity than the meteoric water, groundwater stratification exists in a large portion of the cave. The freshwater layer ranges from 2 centimeters to 5 meters at the surface with the more saline, sulfidic groundwater below (Galdenzi and Maruoka, 2003).

The stratification of water types creates a redox gradient in the aquifer pools as the waters react with each other and cave air. Microbial biofilms utilize the gradients, with populations of sulfur-oxidizing microbes existing near the surface of the aquifers and sulfurreducing, anoxic species existing deeper in cave pools. This chemistry provides niche space for chemosynthetic microbial biofilms in the cave’s streams as well as for acidic populations on the cave walls (Galdenzi and Marouka, 2003; Macalady et al., 2006, 2007, 2008; Galdenzi et al., 2008; Jones et al., 2010).


1.3       Sulfuric Acid Speleogenesis

Sulfuric acid speleogenesis (SAS) occurs when sulfuric acid produced from the complete oxidation of sulfide dissolves calcium carbonate (Principi, 1931; Egemeier, 1973; Macalady et al., 2006, 2007, 2008; Galdenzi et al., 2008; Jones et al., 2010; Tsao, 2014; Hamilton et al., 2015). SAS was once thought to be abiotic and to occur at both above and below the water table (Davis, 2981; Egemeier, 1981; Galdenzi, 1990). Below the water table, sulfide can react with the oxygen within the groundwater to produce sulfuric acid and dissolve limestone. This differs from gypsum replacement that occurs above the water table. As sulfide volatilizes and reacts with atmospheric oxygen, sulfuric acid is formed and reacts with calcium carbonate to create gypsum crusts (Egemeier, 1981; Galdenzi, 2005). As the crusts develop, not only do they grow larger, but they also continue dissolving the limestone due to gypsum having an acidic pH. Eventually, the gypsum crusts become large enough that their weight causes them to detach from the walls and cause the cave to grow.

Although SAS was originally thought to be abiotic, it has been show that sulfuroxidizing microbes greatly contribute to cave development (Vlasceanu, et al., 2000; Engel, et al., 2001; Engel, et al., 2004; Macalady, et al., 2007). In Frasassi, both sulfur reducing and sulfur oxidizing microbes have been identified. Bright, white mats of sulfur-oxidizers (Figure 2) generally function near the perennially microoxic (2-25 µM dissolved O2) aquifer surface and can produce H2SO4 as a by-product of oxidizing hydrogen sulfide (Galdenzi, 2005; Galdenzi and Marouka, 2003).  Sulfur-reducing microbes existing lower in the aquifer increase the amount of available H2S. As more H2S is oxidized, more sulfuric acid is produced and more calcium carbonate limestone is dissolved. Furthermore, microbes can concentrate sulfuric acid when they colonize on cave walls and surfaces, localizing dissolution (Engel, et al., 2004).

Despite abiotic subaqueous calcium carbonate dissolution and sub-aerial gypsum replacement occur at Frasassi (Galdenzi, 1990; Galdenzi and Marouka, 2003; Galdenzi, 2005), microbial sulfur oxidation is likely the catalyst for subaerial SAS and may also increase limestone dissolution rates below the water table (Macalady et al., 2006; Macalady et al., 2007; Macalady et al., 2008; Jones et al., 2008). Contribution towards cave development from abiotic processes and sub-aerial microbial sulfur oxidation is better understood than subaqueous microbial sulfur oxidation, but it is vital to understand metabolic function for individual species’ within these subaqueous biofilms as well as microbial community structure and function. By understanding these microbial biofilms, we may acquire a better understanding of microbial sulfur cycling as well as how early chemosynthetic life on Earth functioned in environments with low oxygen. It may also provide a context in which to understand how life may exist in other habitable zones other than on Earth where redox gradients may exist.


1.4. Microbes in Frasassi

Within Frasassi, microbial life found near the water table is conspicuous, with biofilms being common both above and below the water table (Figure 1.4). Microbial abundance and diversity in the cave have been linked to sulfide to oxygen ratios and flow rates (Jones, et al., 2008) with common taxonomic groups found on both the cave walls and within the aquifer streams and pools (Macalady, et al. 2006; Macalady, et al., 2008). A niche model by Macalady et al., (2008) established that Beggiatoa dominated slow-flow regions regardless of sulfide to oxygen ratios in the bulk stream water, Thiothrix dominated in areas with turbulent flow and low sulfide to oxygen rations, and filamentous Epsilonproteobacteria dominated in areas with turbulent flow and high sulfide to oxygen ratios. Macalady et al. (2008) also showed that Thiobacillus baregensis (“T.bar”)-like populations were abundant throughout cave microbial communities. Investigating T.bar further, Tsao (2014) found that these populations did not correlate with sulfide to oxygen ratios but had genes for sulfur oxidation as well as dissimilatory nitrate reduction genes. These investigations put into question whether or not nitrate was being used in situ since cultures using nitrate grew the fastest. The same occurred for Epsilonproteobacteria cultures, where nitrate allowed for faster growth compared to aerobic cultures (Tsao, 2014).

Because Epsilonproteobacteria are an understudied clade and the only known culture of T.bar was subsequently lost, investigations into their metabolic pathways are of specific interest. Although we understand these populations are oxidizing sulfur, it is not completely understood how or with the use of which electron acceptor. Samples of subaqueous biofilms were previously taken from various sites within Frasassi for metagenomic analysis. These sequence data are used in this thesis to link identified genes to their possible function. By conducting these analyses, we can better understand what metabolic pathways are employed by individual species to oxidize sulfur within these subaqueous biofilms, furthering our knowledge of microbial function on individual and community levels as well as on cave development.








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