Microbial Diversity in Caves

Geomicrobiology Journal

ISSN: 0149-0451 (Print) 1521-0529 (Online) Journal homepage: http://www.tandfonline.com/loi/ugmb20

Microbial Diversity in Caves Karolina Tomczyk-Żak & Urszula Zielenkiewicz To cite this article: Karolina Tomczyk-Żak & Urszula Zielenkiewicz (2015): Microbial Diversity in Caves, Geomicrobiology Journal, DOI: 10.1080/01490451.2014.1003341 To link to this article: http://dx.doi.org/10.1080/01490451.2014.1003341

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Date: 30 November 2015, At: 02:57

Geomicrobiology Journal, (2015) 0, 1–19 Copyright © Taylor & Francis Group, LLC ISSN: 0149-0451 print / 1521-0529 online DOI: 10.1080/01490451.2014.1003341

Microbial Diversity in Caves _ KAROLINA TOMCZYK-ZAK and URSZULA ZIELENKIEWICZ*

Downloaded by [Institute of Biochemistry & Biophysics], [Urszula Zielenkiewicz] at 02:57 30 November 2015

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Received March 2014; Accepted December 2014

Relatively stable physical conditions in caves allow for the examination of the relationship between geochemical processes and the activity of microorganisms, reflected in substantial rock alterations, formation of new structures, surface deterioration and cave expansion. Although caves are considered as extreme environments, they are inhabited by microbial communities with unexpected diversity. While Proteobacteria and Actinobacteria are the most ubiquitous groups, also the presence of Archaea has been frequently noted recently. Here, we present a summary of results on diversity of cave microorganisms in the context of taxon distribution as well as the contribution and role of individual taxa in cave ecosystems. Keywords: biodiversity, biofilm, caves

Introduction Caves are natural geological formations created by empty spaces in the rock, which are considered as extreme environments, unfavourable to the development of life due to severe abiotic conditions. At the same time, they constitute ecological niches for highly specialized microorganisms (Schabereiter-Gurtner et al. 2004). The most common types of caves are karst caves formed from limestone rocks, and caves created by lava cavities in the basalt rock. Caves constitute oligotrophic ecosystems (less than 2 mg of total organic carbon (TOC) per liter), characterized by total darkness or low level of light, low stable temperature and high humidity. Despite oligotrophic conditions, the average number of microorganisms growing in these ecosystems is 106 cells/g of rock (Barton and Jurado 2007). Photosynthetic activity occurs only in places where light has access, usually at the entrance to a cave, but also inside the cave due to the presence of artificial lights mounted for the public. Lack of light inhibits production of primary organic matter by photosynthetic microorganisms. Alternative methods of carbon assimilation are associated with chemoautotrophy. In such conditions, primary organic matter is produced by chemolithoautotrophic microorganisms, which derive energy from binding not only hydrogen, nitrogen, or volatile organic compounds, but also from oxidation of reduced metal ions (e.g., manganese, iron)

*Address correspondence to Urszula Zielenkiewicz, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi nskiego 5A, 02-106 Warsaw, Poland; Email: ulazet@ibb. waw.pl Color versions for one or more of the figures in the article can be found online at www.tandfonline.com/ugmb.

present in the rocks (Gadd 2010; Northup and Lavoie 2001). The main source of allochtonous matter in the cave is water leaking through cracks in the rock from the soil located above, streams depositing sediments of clay on the floor and walls of the cave, or air that carries organic matter particles. Other sources of organic compounds are plant roots or remains of animal and human activity. The presence of organic matter allows for development of heterotrophs. The study of species composition of microbial populations present in oligotrophic cave environments revealed a surprisingly high degree of biodiversity within the Bacteria domain, and the presence of representatives from the Archaea domain. Such high biodiversity, in the environment with nutrient deficiency, is a paradox in terms of ecological principles of competitive exclusion, according to which two species cannot coexist if they utilize the same nutrient source, which is deficient. However, due to the presence of low amounts of nutrients, which are a chemically complex source of carbon and energy, all high-energy reactions necessary for growth are unlikely to be performed by a single microorganism (Barton and Jurado 2007). To overcome these limitations, instead of competing for nutrients, microbial populations form collective structures (different types of biofilms) in which they cooperate and enter into mutualistic relationships. These interactions can explain the lack of growth in laboratory conditions of most of the environmental microorganisms, whose growth depends on specific interactions with other species and cannot grow alone (Grotenhuis et al. 1991; Mohn and Tiedje 1992). With the discovery of mineral deposits, whose formation is difficult to explain taking into account only geological processes, an increased interest in microorganisms and microbial biodiversity of caves in which they occur was noted. Detailed studies have shown that certain calcified structures such as


2

_ and Zielenkiewicz Tomczyk-Zak

Fig. 1. Calcified structures of biogenic origin discovered in caves. (a) - pool fingers (modified after Kristen Bobo, Buffalo Cave, Fentress Co, TN); http://www.flickr.com/photos/chucksutherland/6670733837/sizes/o/in/photostream/. (b) U-loops (modified after Kenneth Ingham© 2004); http://www.caveslime.org/kids/cavejourney/Images/Cave-Journey/uLoop.jpg

“U-loops” and “pool fingers” (Figure 1) are of biogenic origin (Melim et al. 2009). By interacting with minerals microorganisms play an important role in the formation of caves, participating in shaping such structures as stalactites, stalagmites, as well as in decay of rocks (accumulation of metal oxides as a result of microbial activity, in abiotic reactions causing formation of brittle rock surface), which lead to cave expansions. Some microorganisms precipitate CaCO3 on the surface of their cells, which contributes to formation of limestone (Sanchez Moral et al. 2003). Many famous caves that contain Paleolithic paintings, such as Tito Bustillo, Altamira, Llonin and La Garma, are exposed to microbial colonization, which represent a threat to the preserved cave paintings. Microbial colonization of the caves has a destructive influence on the pigments of prehistoric and medieval paintings, and on the bedrock, on which they are situated (Saiz-Jimenez 2011). For this reason, efforts should be made to characterize microbial communities colonizing these caves, in order to develop methods that would effectively prevent their growth. Early studies of the diversity of microbial communities were carried out using traditional culturing techniques. The use of these methods led to a high underestimation of microbial biodiversity due to the inability to culture most of the microorganisms (Torsvik and Ovreas 2002). Molecular techniques allow researchers to bypass this problem and, at the same time, only slightly interfere with the tested environment due to small sample sizes. However, recent studies have shown that despite more accurate methods of determining species composition, organisms that have been cultured from the same sample are often neglected. This is probably due to the fact that strains that grow in vitro generally comprise a small portion of environmental populations (Donachie et al. 2007). Currently, the most common methods for testing microbial diversity are based on analyzing molecular markers, which include small (16S rRNA) and large (23S rRNA) subunits of ribosomal RNA genes, as well as

functional genes such as soxB (active in sulfur-oxidizing microorganisms), amoA (active in ammonia-oxidizing organisms), RuBisCO (gene found in chemoautotrophic microorganisms) and genes critical for cell function, i.e., “housekeeping genes” like rpoB, recA or gyrB (Holmes et al. 2004). Sequences of these genes are amplified from total DNA isolated from environmental samples, and then cloned and sequenced, or the PCR product is separated and analyzed by electrophoresis using “fingerprinting” methods, such as: DGGE (Denaturing Gradient Gel Electrophoresis), RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), RISA (Ribosomal Intergenic Spacer Analysis) and other (Dorigo et al. 2005). Another popular method, called ESS (Environmental Shotgun Sequencing), is based on fragmenting of microbial genomic DNA, which is subsequently introduced into suitable vectors and sequenced. Currently, a second generation of sequencing methods is used, e.g. great popularity was gained by pyrosequencing of amplicons generated by PCR using total DNA collected from a given environment (Wooley et al. 2010). Another frequently applied technique is FISH (Fluorescent In Situ Hybridization) (Moter and G€ obel 2000), which is based on nucleic acid hybridization with fluorescently labeled specific oligonucleotides. This technique allows for simultaneous identification of microorganisms and estimation of their quantity in the sample. SIP analysis (Stable Isotope Probing) is commonly used to test which microorganisms actively assimilate CO2. It is based on incubation of microbial populations with carbon isotope 13C and then isolation of labeled DNA, which serves as a template for amplification of the 16S rRNA gene (Chen et al. 2009). Furthermore, tests are often carried out (at the level of transcription) to determine, which microorganisms, populating a specific ecosystem, are metabolically active. For this purpose, total RNA is isolated, and then functional

Microbial Diversity in Caves


transcripts of specific genes are searched for. This method is usually combined with amplification of 16S rRNA from total RNA as template. For a more complete overview of the environmental biodiversity, several methods are often used simultaneously. A suite of methods allows for a better understanding of what the different microorganisms are doing in the environment functionally.

Microbial Diversity of Caves The occurrence and structure of microbial communities is influenced by such factors as: pH, availability of nutrients, light, oxygen, sulfur and compounds of other metals, humidity, and susceptibility of the substrate to colonization. Alteration of physico-chemical conditions can also influence a change in species composition, for example in the water mats of streams in the Lower Kane Cave, rich in sulfur compounds (Engel et al. 2010; Jones and Bennett 2014). The population structure in these mats is different along the stream due to the changing concentrations of oxygen and sulfide. The water flowing directly from the spring orifices in the cave, containing sulfur at high concentration and small amounts of oxygen, is dominated by e-Proteobacteria. However, the water flowing out of the cavern to the external environment, which contains large amounts of oxygen and low concentrations of sulfur, is dominated by g-Proteobacteria. In contrast to mentioned above, other factors have no so obvious influence on microbial diversity in caves. It seems that the occasional or limited human intervention in the cave environment does not necessarily affect the structure and composition of microbial populations inhabiting these sites. An example is the Herrenberg Cave, a typical karst cave, which was accidently discovered during the digging of a new railway tunnel in the Thuringian Forest in Germany. It differs from other previously studied caves due to the lack of prior presence and interference of animals and humans. Studies on the composition of microbial populations inhabiting this cave did not reveal any unique phylogenetic patterns compared to other cave environments. This may indicate, as the authors themselves suggest, that the microbial communities inhabiting karst cave ecosystems can be stable and insusceptible to low human interference (Rusznyak et al. 2012). However, Adetutu et al. (2012) showed that tourist accessible regions of Naracoorte Caves in Australia had consistently higher bacterial counts than their undisturbed areas. Differences in bacterial diversity in cave sediments had been attributed to the presence of exogenous organic matter of human origin. An interesting example of changes in the microbial biodiversity dependent on subtle alterations in the environment is the limestone Kartchner Caverns (Ortiz et al. 2013). This cave has a humidity of 99.4%, temperature of 19.8 C and a CO2 concentration from 1000 to 5000 ppm. Analysis of the 454-pyrotag of the V6 region of 16S rRNA was used to assess biodiversity. Samples for the study were collected, among other sites, from stalactites in the Big Wall room. Results of the analysis indicated that speleothems in the Big Wall room

3 differed in terms of dominance of particular taxonomic groups in the populations that inhabit them. Three types of microbial communities could be distinguished. Type I (identified in four of the seven stalactites tested), which was characterized by reduced biodiversity, was clearly dominated by Actinobacteria (from 48 to 66% OTUs). The second largest group was Proteobacteria, with a-Proteobacteria as the predominant class. In the second type of community (2 sites of seven), characterized by greater biodiversity, the most numerous microorganisms belonged to Proteobacteria (33–49% OTUs) within which d-Proteobacteria were represented in greater numbers than in type I, lower quantity of a-Proteobacteria were present, and a comparable number of g-Proteobacteria. Actinobacteria were the second largest group (28– 32% OTU). Type III population, identified only on one stalactite, had a unique phylogenetic profile, in spite of the fact that the location of this site was just 1.5 m away from the others. The largest group in this type was Acidobacteria (33% OTU), Proteobacteria were represented only by 14% OTU, and Gemmatimonadets constituted 10% of the population. In the previous two types, Acidobacteria accounted for a maximum of 11% of bacterial populations. The reason for such different structure of the population was likely an increased metal content (2.3- to 173-fold) in this stalactite compared to other stalactites examined. An interesting example of interrelation observed between microbial composition and secondary mineral deposits are lava caves (Northup et al. 2011). In these environments microbial composition is generally similar at the phylum level, in spite of different climate conditions and geographical location of the studied caves. However, significant differences in composition of Actinobacteria, Gemmatimonadetes, Firmicutes and Acidobacteria at the OTU level were observed in yellow and white mats of different lava caves on Hawaii and Azores (Hathaway et al. 2014). There are several papers describing community structures related to different types of caves (Barton and Jurado 2007; Barton et al. 2007; Engel 2010; Lee et al. 2012). In this work, we will present microbial biodiversity of caves as well as participation and the role of individual taxa in these ecosystems. Although caves are colonized by many different organisms, this review is focused only on prokaryotic microbes often organized in biofilm communities. Table 1 demonstrates the biodiversity of caves based on the presence of taxonomic groups.

Groups of Bacteria Frequently Present in Caves Proteobacteria Proteobacteria are a cosmopolitan group of bacteria that is prevalent and abundant in caves. In the Spanish Altamira Cave, which is one of the most studied caves due to the famous Palaeolithic paintings, Proteobacteria are dominant in the water dripping from the walls (Laiz et al. 1999; Schabereiter-Gurtner et al. 2002a) as well as in the aggregations colonizing rocks (Portillo et al. 2008, 2009). Microorganisms belonging to this group also dominate the rocks in caves Llonin, La Garma (Schabereiter-Gurtner et al. 2004), Tito

4

>CPresence of defined group; CCRelative high abundance

Altamira (Portillo et al. 2008, 2009; Schabereiter-Gurtner et al. 2002a) Tito Bustillo (SchabereiterGurtner et al. 2002b) Llonin, La Garma (SchabereiterGurtner et al. 2004) Lechuigilla, Spider ( Northup et al. 2003) Pajsarjeva jama (Pasic et al. 2010) Niu Cave (Zhou et al. 2007) Wind Cave (Chelius et al. 2004) Kartchner Caverns (Ikner et al. 2007) Cave of Bats (Urzì et al. 2010) Carlsbad Cavern (Barton et al. 2007) Barenschacht (Shabarova and Pernthaler 2010) Herrenberg (Rusznyak et al. 2012) Stalactites Fluvial sediments Naracoorte Caves (Adetutu et al. 2012) Tourists accessible areas Areas inaccessible to tourists Magura Cave (Tomova et al. 2012) Cave of Crystals (Quintana et al. 2013) Carter Saltpeter Cave (Carmichael et al. 2013) Weebubbie cave (Tetu et al. 2013) Hawaiian, Azorean, New Mexico lava caves (Hathaway et al. 2014; Northup et al. 2011) Sulfur caves Lower Kane (Engel et al. 2003, 2004, 2010) Movile (Chen et al. 2009) Grotta Sulfurea Frasassi (Macalady et al. 2006) Grotta del Fiume Frasassi (Macalady et al. 2007) Grotta Nuova di Rio Garrafo (Jones et al. 2010)

Caves (reference)

Taxonomic group

C C C C C CC CC CC

CC C

CC

CC

C

CC

C

C CC

C

C C

CC CC CC

CC

CC CC C C

CC

C

C

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C

C

C

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CC C

C

C CC

C

CC CC

CC

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C C

C

CC

C C

C

CC

C

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C

C

C C

C C

C CC

C

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CC

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C

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CC

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C

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C C C

C C

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C C

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CC CC

C

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C C

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C CC

C

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C C

C

CC

C

CC

C

C

C

C

CC CC C

C C

C

CC

CC

C

C

C C

C

C

C

C C

C

C

C

C

C C

C

C

C

C

C C

C

CC C

C

C

CC

C

CC

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C

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C

C

C

C

C

C

C

C

C

C

C

C

C

CC

C

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CC

C C

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C

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a-Proteo- b-Proteo- g-Proteo- d-Proteo- e-ProteoActino- Acido- BacteroVerruco- PlanctoGemmatiCyanoFibrobacteria bacteria bacteria bacteria bacteria Nitrospirae bacteria bacteria idetes Firmicutes microbia mycetes Chloroflexi monadetes Spirochetes bacteria Chlorobi bacteres Archaea

Table 1. Microbial diversity of taxonomic groups in caves



Microbial Diversity in Caves Bustillo (Schabereiter-Gurtner et al. 2002b), Pajsarjeva jama (Pasic et al. 2010), as well as in microbial mats on basalt walls of lava caves (Hathaway et al. 2014; Northup et al. 2011), on the stalactites of the Herrenberg Cave (Rusznyak et al. 2012), in the soil of the Niu cave (Zhou et al. 2007), sediments of the Wind Cave (Chelius and Moore 2004) and in pools of the Barenschacht Cave (Shabarova and Pernthaler 2010). Proteobacteria as the largest taxonomic group can also be found in extreme environments, such as sulfurous caves, e.g., Grotta Nuova di Rio Garrafo (Jones et al. 2010), Parker (Angert et al. 1998), Cesspool (Engel et al. 2001), Lower Kane (Engel et al. 2003), Movile (Sarbu 2000). In Grotta Sulfurea of the Frasassi cave system, Proteobacteria are involved in the sulfur cycle and represent >75% of the clone library created from the 16S rRNA genes of the water mat organisms (Macalady et al. 2006). Moreover, they are the dominant part of the biodiversity in the biofilm, hanging from the walls of the cave with an extremely acidic pH (0–1), found in the Grotta del Fiume (Macalady et al. 2007). Success of Proteobacteria colonization may be attributed in part to their ability to degrade a wide range of organic compounds. Studies have shown that increased availability of nutrients, which may inherently be associated with the presence of animals and humans in caves, may explain the dominance of Proteobacteria in the microbial population. Ikner and others (2007) compared the biodiversity of microorganisms tested at three locations of the Kartchner Caverns. Samples were collected from the rock of the hall visited by tourists: frequently (200,000 per year), occasionally (30– 40 years) and rarely (2–3 people per year). Proteobacteria dominated (77% of cultured isolates) in the site most frequently visited by tourists, while the other two areas were dominated by microorganisms of Firmicutes type (66% and 52% of isolates from rarely and occasionally visited sites, respectively). Northup et al. (2003) compared the microbial diversity of iron-manganese deposits in the extremely oligotrophic Lechuguilla Cave not visited by tourists to that of the ironmanganese deposits from the shallow Spider Cave explored by tourists. Researchers have shown an increase in nitrogenfixing Proteobacteria in the Spider Cave. Other studies (Spilde et al. 2005) revealed that Proteobacteria contribute to the formation of ferromanganese deposits. In the highly frequented Lascaux Cave, microbial populations consisted almost entirely of Proteobacteria (Bastian at al. 2009). Despite the undeniable dominance of Proteobacteria in cave ecosystems, representation of particular classes of this type varied in different environments. a-Proteobacteria This group is metabolically, morphologically and ecologically diverse. It consists of organisms such as chemoorganotrophs, chemolithotrophs and facultative photoheterotrophs, which are aerobic or facultatively anaerobic. Typically, a-Proteobacteria are surface microorganisms, usually constituting a small part of microbial populations inhabiting the caves. However, in some cave ecosystems they are one of the most abundant taxa, e.g., in the caves containing Palaeolithic

5 paintings. Within this class, Rhizobiaceae are strongly represented in Lloni and La Garma (Schabereiter-Gurtner et al. 2004) caves, as well as Altamira and Tito Bustillo (Schabereiter-Gurtner et al. 2002a, 2002b). Lloni and La Garma, which possess ecosystems inaccessible to tourists, maintain a constant temperature of about 13 C and 100% humidity. Altamira and Tito Bustillo caves have similar physical conditions, but are often visited by tourists. Despite this fundamental difference, the microbial populations of Tito Bustillo and La Garma do not differ in any significant way. This may indicate that the microorganisms identified in both sites were typical bacteria for these environments, and the anthropologic impact was low or negligible (Schabereiter-Gurtner et al. 2004). The best-studied cave with Paleolithic paintings in terms of species composition is the Altamira Cave. In this cave, researchers observed three morphologically distinct aggregations (white, gray and yellow), consisting of numerous microorganisms of different taxonomic groups (Portillo et al. 2008; Portillo et al. 2009). In the first two types, a-Proteobacteria were the most abundant class, but differed in the number of metabolically active microorganisms. Metabolically active bacteria comprised a small fraction of white colonies (around 6% of the 16S rRNA sequences from total RNA) and a large part of gray aggregations (35.7% of the sequences), and was poorly represented in yellow colonies. In the population of microorganisms growing on ironmanganese deposits of Lechuguilla and Spider caves (air temperature » 19 C, relative humidity –; 100%, pH » 8), a-Proteobacteria were also one of the most numerous groups (second group in terms of size; » 17% of the 16S rRNA sequences) (Northup et al. 2003). Sequence analyses showed highest similarity to Afipia spp., Mesorhizobium spp. and Hyphomicrobium spp. The latter bacterium is capable of manganese oxidation. By contrast, in the soil environment of the Niu Cave, a-Proteobacteria were the third largest group of microorganisms (11.2%) (Zhou et al. 2007). Most of the 16S rRNA sequences have been attributed to orders: Rhizobiales, Sphingomonadales and Rhodospirales. Among them, Mesorhizobium spp. was identified to have the ability to fix nitrogen (Bottomley 1992), and Pedomicrobium spp. to oxidize iron and manganese. In Hawaiian and Azorean lava caves (air temperature 14–19 C for Hawaiian and 15–16 C for Azorean, relative humidity approx. 95%), a-Proteobacteria presented a second most numerous group in population (13% and 15%, respectively) of yellow and white mats (Hathaway et al. 2014). a-Proteobacteria were one of the most numerous classes (together with g-Proteobacteria) also in the consortium of microorganisms growing in the Carlsbad Cavern (Barton et al. 2007), on a chemically complex rock, containing hematite, magnetite, tourmaline, zircon, rutile, apatite and epidote. The density of the organic matter of this rock is low (»3 mg/ g¡1). The class of a–Proteobacteria was most strongly represented by genus Brevundimonas. Bacteria belonging to this genus are oligotrophic organisms able to adapt to ecosystems extremely poor in nutrients (Dworkin 2002; Li et al. 2004). In the same cave, but on a different rock type composed primarily of CaCO3, a-Proteobacteria were not so common, in


6 spite of higher density of organic matter. A small part of this class was also found in microbial populations of sediments in the Wind Cave (Chelius and Moore 2004), water mats of sulphur rich caves: Lower Kane (Engel et al. 2004; 2010) and Movile (Chen et al. 2009); rocks in Pajsarjeva jama (Pasic et al. 2010), Kartchner (Ikner et al. 2007), Cave of Bats (Urzì et al. 2010), stalactites and fluvial sediments of the Herrenberg Cave (Rusznyak et al. 2012), ferromanganese deposits of the Carter Saltpeter Cave (Carmichael et al. 2013), slimes in Weebubbie Cave (Tetu et al. 2013) and the Barenschacht Cave pools (Shabarova and Pernthaler 2010). b-Proteobacteria Microorganisms of this class are phenotypically, metabolically and ecologically diverse. Among bacteria constituting this class are obligate aerobes and facultative anaerobes, chemoorganotrophs as well as obligatory or facultative chemolithotrophs. These microorganisms are dominant in the water mat populations of the Movile Cave (Chen et al. 2009), which is an isolated cavern, characterized by neutral pH, temperature of 20 C, high concentration of reduced sulfur compounds, ammonium and methane in water, and the presence of methane, hydrogen sulfide and CO2 in the atmosphere. In this ecosystem, most common are chemolithoautotrophic processes, and the main primary producers are microorganisms oxidizing sulfur, ammonia and nitrites. Studies based on examination of 16S rRNA genes as well as functional genes demonstrated the presence of microorganisms of the following taxonomic classes: sulfur-oxidizing bacteria, Thiobacillus spp., Thiobacter spp., methylotrophs, Methylotenera spp., Methylophilus spp., Methylovorus spp. and Methylibium petroleiphilum (facultative methylotrophs), denitrifying bacteria, Denitratisoma spp., ammonia-oxidizing bacteria, Nitrosomonas europea, Nitrosomonas oligotropa (dominant species), nitrite-oxidizing bacteria, Nitrotoga arctica. The genus Nitrosomonas spp. constitutes probably the main group of nitrifying microorganisms in the Movile Cave. The cave is characterized by significant fluctuations in the concentration of methane. Most likely methylotrophs use methanol produced by methanotrophs, which were also identified in this cave (Hutchens et al. 2004). SIP analysis detected microorganisms actively assimilating CO2, i.e., Thiobacillus spp., Thiobacter spp., Nitrosomonas spp. and Nitrotoga spp. In the Herrenberg Cave, b-Proteobacteria were the second largest class of microorganisms inhabiting stalactites of this cave (they accounted for about 20% of all 16S rRNA sequences) (Rusznyak et al. 2012). Herrenberg Cave is a karst cave with a low temperature of 7–9 C, which has not been contaminated by human or animal presence in the past. In the case of water mats of the Lower Kane Cave, b-Proteobacteria were the third largest taxonomic group of identified microbial organisms (they constituted 11.7% of the population) (Engel et al. 2004). This cave has a high concentration of sulfur, a stable temperature of 22 C and pH of 7.2. Thiobacillus spp. detected in mats (facultative chemolithoautotroph oxidizing sulfur) were more numerous in areas with lower concentration of sulfur and high oxygen concentration.

_ and Zielenkiewicz Tomczyk-Zak b-Proteobacteria dominated in the pool of the epiphreatic zone (periodically flooded with water) and two pools of the vadose zone (not flooded, only percolating water) of the Barenschacht Cave (Shabarova and Pernthaler 2010). The temperature of the cave ranges from 6.4 to 6.8 C, while pH is » 7. The content of organic matter in the vadose zone pools was low and in the epiphreatic pool was 12 times higher (1.9 to 4.5 mg L¡1). All 3 pools were highly diverse. Microbial population of the epiphreatic zone pool consisted of microorganisms collected by water from a variety of aerobic and anaerobic niches of the cave and soil. This site was dominated by the b-Proteobacteria genus - Methylotenera. Bacteria of this type are capable of utilizing methylamine as a sole carbon source under aerobic and microaerophilic conditions (Kalyuzhnaya et al. 2006). They are not typical for this ecosystem and most likely have been brought in with water. In the other two pools of the vadose zone, Oxalobacteraceae constituted a numerous family. The only common genus for all three pools was Rhodoferax, often occurring in ground waters, but also identified on the surface of cave rocks (Barton et al. 2007). The study of two populations growing on chemically complex rock in Carlsbad Cavern by Barton et al. (2007), revealed that b-Proteobacteria were numerous, with the most abundant genus being Massilia. This genus is composed of bacteria species that utilize complex carbohydrates and other organic compounds as energy and carbon source with simultaneous production of acids (Dworkin 2002). Such activity may explain the structural changes observed in the rock, where the calcareous sandstone binders are readily dissolved by acid, which is corrosive to the rock. The presence of Herbaspirillum frisingense and Janthinobacterium agaricidamnosusm is indicative of nitrogen assimilation in this ecosystem. In contrast to chemically complex rocks, the limestone rock in the Carlsbad Cavern is composed mainly of CaCO3. In this environment b-Proteobacteria were sparse, represented by Comamonas spp., a saprophyte, degrading aromatic nitrogen-containing compounds, with the simultaneous liberation of ammonia and ammonium nitrate. This bacterium is usually found in environments containing low amount of nutrients (Dworkin 2002). b-Proteobacteria were the dominant class of Proteobacteria in sites of the Kartchner Cave intensely visited by tourists (Ikner et al. 2007). The cave has a constant temperature of 19.8 C and humidity at the level of 99.4%. By contrast, in Lechuguilla and Spider caves this class constituted 14% of the 16S rRNA sequences. These sequences showed the highest similarity to Aquaspirillum spp. and Variovorax spp. (aerobic chemoorganotrophs). Some strains of those genera are capable of chemolithoautotrophic use of hydrogen as an energy source. Variovorax spp. cells can incorporate rare earth elements, such as minerals, neodymium, cerium and lanthanum, contained in the iron-manganese deposits of both caverns (Northup et al. 2003). b-Proteobacteria were the second-most numerous group in ferromanganese deposits of the Carter Saltpeter Cave (Carmichael et al. 2013). This group was represented by cultivated Mn(II) oxidizing isolates: Janthinobacterium sp A6. and Leptothrix sp. G6. Carmichael et al. showed that the


Microbial Diversity in Caves isolated Leptothrix was able to carry out both Mn and Fe oxidation, confirming the role of this bacteria in the formation of ferromanganese deposits in the cave. In both white and yellow microbial mats, as well as secondary mineral deposits of Hawaiian, Azorean, and New Mexico lava caves, b-Proteobacteria were moderately significant part of the whole population of microbes (Hathaway et al. 2014; Northup et al. 2011). In gray, yellow and white colonies of the Altamira Cave, b-Proteobacteria were metabolically active, but were not strongly represented. The most numerous metabolically active microorganisms belonged to the following orders: Nitrosomonadales (involved in the nitrogen cycle), Burkholderiales and Rhodocyclaes (sulfur-reducing bacteria Thauera spp.). A limited number of b-Proteobacteria was also present in such caves as: the Wind Cave (Chelius and Moore 2004), Tito Bustillo (Schabereiter-Gurtner et al. 2002b), Lloni and LaGrarma (Schabereiter-Gurtner et al. 2004), Pajsarjeva jama (Pasic et al. 2010) Grotta del Fiume and the Grotta Sulfurea Frassasi cave system (Macalady et al. 2006; 2007), Niu (Zhou et al. 2007), Magura Cave (Tomova et al. 2012), Weebubbie Cave (Tetu et al. 2013) and fluvial sediments of the Herrenberg Cave (Rusznyak et al. 2012). g-Proteobacteria g-Proteobacteria are an important part of biofilms developing in cave waters rich in sulfur. They also inhabit cave rocks. In the biofilm of the Cesspool Cave developing in a stream containing high sulfur concentration about half of the 16S rRNA sequences analyzed were assigned to the group of g-Proteobacteria (Engel et al. 2001). In the Movile Cave this group was the second largest taxonomic class. Organisms that have been identified belonged to the: Xanthomonadales family, sulfur-oxidizing bacteria and representatives of genera Thiovigra, Thiothrix, Thioploca, Halothiobacillus and Beggiatoa. Additionally, several microorganisms associated only at the family level to Thiotrichaceae have been found (Chen et al. 2009). Unclassified Thiotrichaceae and microorganisms of the genus Thioploca actively assimilated CO2 in SIP analysis. In the Grotta Sulfurea, the g-Proteobacteria group represented the largest part of the microbial population (Macalady et al. 2006). Grotta Sulfurea, part of the Frasassi cave system, is a rare example of an actively forming sulfide cave, in which as a result of abiotic and biotic factors sulfuric acid that is

7 produced reacts with calcium carbonate from rocks. This, in turn, leads to an intensive corrosion of the limestone (15 mg of CaCO3/cm2/year). Such environment is based on the sulfur cycle, isolated from carbon and nitrogen sources. The temperature at this location is 13.6 C and pH of the water where mats develop is 7.3. Water mats are morphologically distinguishable and defined as cottony water mats (in the calm waters) or feathery mats (turbulent waters) (Figure 2). The group of g-Proteobacteria was dominant in the cottontype biofilm. The dominance of this group in this type of biofilm is likely related to high concentration of dissolved oxygen, limiting the growth of e-Proteobacteria. Identified 16S rRNA sequences were attributed principally to the genus Beggiatoa (cotton biofilm) and Thiothrix (feathered biofilm). Beggiatoa spp. are facultative anaerobes oxidizing sulfur that colonizes hydrogen sulfide and oxygen gradients. This species was able to grow in a lower concentration of soluble oxygen than Thiothrix spp. (1–2.5 mM) (Nelson et al. 1986). Microorganisms of the genus Thiothrix (also facultative sulfur-oxidizing anaerobes) were observed in areas of the biofilm where the concentration of soluble oxygen exceeded 10 mM (Engel et al. 2004). These bacteria were also able to accumulate sulfur within the cell (Howarth et al. 1999). Results of previous studies suggested that the availability of oxygen is an important factor in controlling the spatial distribution of the two genera. The rich in sulfur Grotta Nuova di Rio Garrafo has neutral pH and high water temperature (50 C). The g-Proteobacteria class constituted the second largest group of bacterial biofilms examined (more than 10% of the population) in this environment (Jones et al. 2010). Among them, sulfur-oxidizing lithothrophic bacteria were identified, e.g. Candidatus Thiobacillus baregensis, Thiofaba tepidiphila and Thiovirga sulfuroxydans. Also among bacterial populations inhabiting stalactites of the karst Herrenberg Cave, the g-Proteobacteria class was the most numerous the 16S rRNA sequences of this class represented about 30% of all sequences obtained (Rusznyak et al. 2012). In slime populations of Weebubbie Cave (high level of sulfates, nitrates and nitrites), g-Proteobacteria were also one of the most numerous groups with Pseudoalteromonas being the most abundant genus (Tetu et al. 2013). Although it is not the most diverse taxonomic class in terms of species composition, g-Proteobacteria occurred most frequently in the extremely acidic (pH 0-1), rich in sulfur compounds and poor in iron ions, Grotta del Fiume of the

Fig. 2. Example of cave water biofilms. A: cottony water mats, B: feathery mats (modified after Macalady et al. 2006).


8 Frasassi cave (air and water temperature is 1–2 C). Dripping biofilms (called snottites) in the Grotta del Fiume are considered one of the least diverse natural biofilms (composed of only six prokaryotic phylotypes) (Baker and Banfield 2003). All sequences of g-Proteobacteria class represented the Acidithiobacillus type. Studies carried out in acidic environments showed a correlation between pH and the domination of Acidithiobacillus (Golyshina and Timmis 2005). Microorganisms of this genus are autotrophs oxidizing reduced sulfur compounds, including thiosulphate, hydrogen sulfide and elemental sulfur. In water mats of the Lower Kane Cave, g-Proteobacteria accounted for 12.2% of the population (the second-most numerous taxonomic group) (Engel et al. 2004). The majority of observed bacteria belonged to the genera Thiotrix and Beggiatoa. It was noted that Thiotrix spp. were present preferentially (were more numerous) in sites with lower concentration of sulfur and high concentration of oxygen – conditions existing closer to the exit of the cave. It is also found in other sulfurous caves, e.g., the Parker Cave (Angert et al. 1998), underwater caves in Florida (Brigmon et al. 2003) or the Cesspool Cave (Engel et al. 2001). In one of the tested mats of the Lower Kane Cave, Enterobacteriaceae with predomionance of Pantoea spp. and Serratia spp. were observed. g-Proteobacteria are the second largest group of microorganisms in the sediments of the Wind Cave (Chelius and Moore 2004). It is a deep cave with neutral pH and a temperature of 13 C, containing a small amount of organic matter, high concentrations of nitrites, nitrates, phosphates, and sodium. Sequences of the 16S rRNA clone library of the Wind Cave showed similarity to the genus Pseudomonas, comprising manganese-oxidizing species. In the chemically complex rocks of the Carlsbad Cavern, g-Proteobacteria were also one of the most numerous classes (Barton et al. 2007). Among this class the most abundantly represented genera were Stenotrophomonas and Delfitia. Bacteria of these genera carry out the denitrification reaction, in which nitrous oxide is formed from ammonia (Dworkin 2002), and the denitrification reaction of complex organic compounds, such as nitrobenzene. In the population of bacteria that inhabited the rock composed of CaCO3 of the Carlsbad Cavern, 16S rRNA sequences of g-Proteobacteria were represented at an average level in comparison to other groups. From the pool of 16S rRNA sequences, Acinetobacter johnsonii was identified as a saprophitic microorganism capable of mobilizing phosphates from inorganic compounds. This bacterium is usually found in an environment containing low amount of nutrients (Dworkin 2002). g-Proteobacteria dominated also the community of the Pajsarjeva jama as 31.5% of all sequences belonged to this class (Pasic et al. 2010). The temperature in the cave is 12 C, humidity 100%, and pH neutral. Most of the 16S rRNA sequences showed strong similarity to 16S rDNA of microorganisms oxidizing hydrogen sulfide. Xathomonadales was another group identified in this cave, as well as in the Altamira cave (Portillo et al. 2008), and due to the production of carotenoids its presence is often correlated with the yellow coloration of the colonies. In the Altamira Cave,

_ and Zielenkiewicz Tomczyk-Zak g-Proteobacteria were the most numerous metabolically active group in both yellow and white colonies (34.2% and 47.9% of 16S rRNA sequences from total RNA, respectively). In addition to the strong representation of Pseudomonadales (Azotobacter spp., Pseudomonas spp.) and the presence of carotenoid-producing Chromatiales and Xanthomonadales, bacteria involved in the nitrogen cycle belonging to Nitrosococcus spp. as well as orders of Enterobacteriales (Escherichia spp.) and Methylococcales (Methylocaldum spp.) were also identified. g-Proteobacteria constituted a small part of the metabolically active population of gray colonies (14.3%) (Portillo et al. 2008; 2009). The most numerous bacterial isolates affiliated to g-Proteobacteria were also in Magura Cave (Tomova et al. 2012). They belong to six genera: Acinetobacter, Enterobacter, Pseudomonas, Obesumbacterium, Serratia and Stenotrophomonas. The authors speculate that domination of Serratia and Pseudomonas could be a result of high level of contamination of cave surfaces probably from insects and bats. In the soil of the Niu Cave (Zhou et al. 2007) and rocks of the La Garma Cave (Schabereiter-Gurtner et al. 2004), Lechuguilla and Spider caves (Northup et al. 2003), g-Proteobacteria microorganisms were one of the most dominant groups. In the Lechuguilla and Spider caves, this group was represented by Pantoea spp. and Stenotrophomonas spp. Pantoea is involved in Fe3C reduction and the nitrogen cycle based on their ability to fix atmospheric nitrogen in the presence of nitrate nitrogen. In Hawaiian, Azorean and New Mexico lava caves g-Proteobacteria were represented by moderately numerous microorganisms (Hathaway et al. 2014; Northup et al. 2011). g-Proteobacteria were found sporadically in caves Barenschacht (Shabarova and Pernthaler 2010), Cave of Bats (Urzì et al. 2010), Karthner (Ikner et al. 2007), Tito Bustillo (Schabereiter-Gurtner et al. 2002b), Carter Saltpeter Cave (Carmichael et al. 2013), and fluvial sediment populations in the Herrenberg Cave (Rusznyak et al. 2012). d-Proteobacteria The majority of microorganisms belonging to the class of d-Proteobacteria are anaerobic sulfate-reducing organisms, able to disproportionate sulfur compounds. In contrast to other tested caves containing high concentration of sulfur compounds, in which d-Proteobacteria were absent or represented a small part of the microbial population (e.g., the Cesspool Cave (Engel et al. 2001); Parker Cave (Angert et al. 1998); Lowe Kane Cave (Engel et al. 2004)), in Grotta Sulfurea, this group was abundant. In the “cotton”-type water mats present in the stream of the Frasassi cave, d-Proteobacteria was the second largest group of microorganisms, while in the feathery mats it was the dominant group. Almost half of the 16S rRNA sequences were attributed to microorganisms of genera Desulfocapsa and Desulfonema. In addition, the Geobacteraceae family was also identified along with species of the following genera: Syntrophobacter, Syntrophus, Desulfoarculum, Desulfobacter and Desulfomonile. Desulfocapsa is an autotrophic microorganism requiring low


Microbial Diversity in Caves sulfide concentrations for efficient growth. It is able to disproportionate sulfites, elemental sulfur and thiosulfates (Janssen et al. 1996) as well as to reduce sulfur and sulfates (Frederiksen and Finster 2004). The Desulfonema genera comprise sulfur-reducing bacteria that may grow as embionts on filaments of the sulfur-oxidizing bacteria, such as Thioploca (Fukui et al. 1999). The white water mats of the sulfur-rich Lower Kane Cave, contained d-Proteobacteria as a small part of the population. 16S rRNA sequences showed a high degree of similarity to bacteria Desulfocapsa thiozymogenes. In the same cave, Engel et al. (2010) identified d-Proteobacteria as the dominant group in the gray water mats present, which are characterized by anaerobic conditions. Among microorganisms identified in this class, sulfur-reducing Desufocapsa spp., Desulfomonile spp. and Chondromyces spp. were found (Engel et al. 2010). d-Proteobacteria were also frequently observed in the Wind Cave sediments (Chelius and Moore 2004). It was the third most dominant group of microorganisms in this cave, similarly as in the case of metabolically active bacteria in the gray and yellow aggregations in the Altamira Cave. The most abundant and active genus in the yellow colonies was Desulfovibrio (obligatory anaerobic sulfur-reducing bacteria), while in gray colonies - Myxococcales. The presence of strict anaerobes in the colony population was possible due to the multilayer colonization, which resulted in the formation of anaerobic microenvironments. The existence of such micro-niches allows microorganisms to carry out redox reactions that have a negative effect on the pigment of cave paintings. In white aggregations, d-Proteobacteria were sparse. In lava caves d-Proteobacteria were represented by moderately numerous groups (Hathaway et al. 2014; Northup et al. 2011). Bacteria belonging to d-Proteobacteria were also identified in populations of Grotta Nuova di Rio Garrafo (Jones et al. 2010), the Herrenberg (Rusznyak et al. 2012), Niu (Zhou et al. 2007), Carter Saltpeter (Carmichael et al. 2013), Weebubbie (Tetu et al. 2013) and Pajsarjeva jama (Pasic et al. 2010) caves, however, they were scarcely represented. e-Proteobacteria The e-Proteobacteria are the least known class of the phylum. This class contains metabolically and ecologically diverse chemolithoautotrophs, microaerophils and anaerobes, which are able to use a variety of inorganic compounds as electron sources. Many of them are thermophiles associated with geothermal vents. Some are important opportunistic pathogens. Microorganisms belonging to e-Proteobacteria are an important, frequently encountered, part of microbial populations in cave waters high in sulfur. Recent studies on sulfuroxidizing microorganisms in the Frasassi Cave suggested that e-Proteobacteria may dominate the population, when the ratio of sulfur to oxygen is high (Macalady et al. 2006, 2008). e-Proteobacteria, which were found in the “feathery” biofilm of the Grotta Sulfurea were the second largest taxonomic group. In most cases, 16S rRNA sequences have been assigned to the genus Arcobacter and other sulfur-oxidizing microorganisms. In the “cotton” biofilm, with high concentration of dissolved oxygen, this group comprised a smaller

9 fraction of the population, about 15% of all microorganisms detected. Organisms belonging to e-Proteobacteria constituted a small part of the population of the rich in sulfur Movile Cave (Chen et al. 2009). Among identified bacteria were also Sulfuricurvum spp., which have the ability to oxidize sulfur, and Desulfobulbaceae spp. that carry out the sulfur reduction reaction. The low number of e-Proteobacteria distinguishes the Movile Cave from other cave ecosystems, which are based on chemoautotrophy, e.g. the Lower Kane Cave, where biodiversity of the microbial population in white water mats is low, but primarily dominated by e-Proteobacteria (68% of the sequences) (Engel et al. 2004). e-Proteobacteria present in the tested water mats of the Lower Kane Cave are involved in anaerobic oxidation of sulfur and are the main producers of chemolithoautotrophic carbon and other nutrients in this poor environment. Studies have shown a sharp decrease in the concentration of dissolved sulfide along the streams, which probably is related mainly to the activity of e-Proteobacteria. It was also noted that together with the change in oxygen and sulfide concentration over the entire length of the streams, the structure of the biofilm population was also altered. e-Proteobacteria were dominant at the opening of the cave, where the rich in sulfides water flowed out, and contained undetectable amounts of oxygen. Closer to the exit of the cave, sulfur and oxygen concentrations were changing, causing a decrease in the number of e-Proteobacteria and more abundant occurrence of bacteria of other taxonomic groups. There is no direct evidence that e-Proteobacteria accumulate sulfur intracellularly as Thiothrix spp., however, there have been cases where microorganisms of this class produced sulfur as a final metabolic product in an environment with low or lack of nitrates (Gevertz et al. 2000; Nemati et al. 2001). On the basis of these results, Engel et al. (2004) assumed that the white color of mats dominated by e-Proteobacteria may be associated with the presence of extracellular sulfur or accumulation of sulfur as a result of nitrate reduction. A recent study conducted by Engel et al. (2010) on the gray anaerobic mats of the Lower Kane Cave demonstrated that in contrast to the white mats of this cave, e-Proteobacteria were numerous, but not a dominant part of the population. Most likely, this group of microorganisms in these mats was involved in anaerobic metabolism of nitrates reduction coupled to the oxidation of sulfide. Dominance of e-Proteobacteria in the examined mats might be associated with a rapid metabolic differentiation during colonization and formation of micro-niches that may be colonized by other bacteria in the following stages (Lopez-Garcia et al. 2003). At the same time, this most metabolically diverse class was the dominant group, limiting the increase of bacteria of other taxonomic classes (Chesson et al. 2002). The class of e-Proteobacteria was also numerous in other cave streams containing high concentrations of sulfur compounds. More than half (73%) of identified 16S rRNA sequences from the clone library created from the Parker Cave stream biofilm was attributed to Thiomicrospira denitrificans (Angert et al. 1998). The Cesspool Cave is similar in this aspect as about half of the sequences (47%) of the 16S


10 rRNA clone library of the biofilm growing in the stream was assigned to e-Proteobacteria (Engel et al. 2001). Another example of a sulfuric cave ecosystem dominated by microorganisms of this taxonomic class is the Grotta Nuova di Rio Garrafo at neutral pH and thermal waters of 35–50 C (Jones et al. 2010). In this cave e-Proteobacteria accounted for over 46% of the population of bacterial biofilms. This group was most abundantly represented by Sulfurovumales, which sequences comprised more than 40% of the 16S rRNA genes identified. These bacteria are either mixo- or litho-trophic with the ability to oxidize and reduce sulfur compounds, often occurring in populations inhabiting sulfur caves and streams around the world (Shabarova and Pernthaler 2010). Rossmassler et al. (2012) showed that environmental variables influence community composition in geographically separated sulfidic caves dominated by e-Proteobacteria. While some distantly located communities (>8000 km) shared closely related populations, those from other sites (with higher temperatures and intermediate concentrations of dissolved oxygen) had nearly clonal populations, phylogenetically distinct. The class of e-Proteobacteria was also identified in all tested cave pools of the Barenschacht Cave (Shabarova and Pernthaler 2010), but it constituted an insignificant portion of the population. Nitrospirae The phylum Nitrospirae contains only a few genera of Gramnegative bacteria: Nitrospira, Leptospirillum, Thermodesulfovibrio and Candidata Magnetobacterium and Magnetoovum, with no obvious phylogenetic relations (Kunisawa 2010). Representatives of Nitrospirae are often present in aquatic environments: ground, lake and river waters, as well as hydrothermal fields and wastewater treatment systems. Known microorganisms are metabolically diverse, aerobic, mandatory chemolithoautotrophs, which derive energy for growth from the oxidation of nitrites or iron, as well as have the ability for anaerobic reduction of sulfates (Pohlman et al. 1997). Several magnetotactic members of this phylum have been described in culture-independent studies (Jogler et al. 2010; Lefevre et al. 2011). This taxonomic type is often encountered in cave ecosystems, but very few microorganisms belonging to this group have been characterized to date. Nitrospirae is a commonly encountered type in the Lechuguilla Cave (16% of 16S rRNA sequences, the third most numerous group). Sequences presented highest homology with Leptospirillum ferrooxidanse and Nitrospira marina (Northup et al. 2003). Nitrospira spp. are aerobic chemolithoautotrophs, oxidating nitrates, capable of mixotrophic growth. L. ferrooxidans is a microorganism oxidizing iron, widely present in mines with acidic pH (Schrenk et al. 1998). The relatively high proportion of microorganisms belonging to the Nitrospira type (15.2%) was also found in the Pajsarjeva jama (Pasic et al. 2010). The Nitrospirae was relatively moderately represented group in lava caves (Hathaway et al. 2014; Northup et al. 2011). The low number of Nitrospirae-type bacteria was found in the following caves: Lower Kane (Engel et al. 2010), Niu

_ and Zielenkiewicz Tomczyk-Zak (Zhou et al. 2007), Barenschacht (Shabarova and Pernthaler 2010), La Garma, Llonin, Tito Bustillo (Schabereiter-Gurtner et al. 2002b, 2004), Altamira (Portillo et al. 2008), Movile (Chen et al. 2009), Weebubbie (Tetu et al. 2013) and Herrenberg (Rusznyak et al. 2012).

Actinobacteria Microorganisms of Actinobacteria type are Gram-positive bacteria with a high metabolic potential, which DNA is rich in GC base pairs, frequently dominating in soils. They often colonize the rock walls of caves, as well as crystal cave formations such as stalactites and stalagmites. They are known for the production of various types of crystals found in microbial cave populations. For this reason, it is believed that this group of microorganisms is involved in biomineralization processes in these ecosystems (Barton et al. 2001; Canaveras et al. 2001; Groth et al. 2001; Jones 2001; Laiz et al. 1999). Studies on biodiversity conducted with the use of culturing techniques often revealed the dominance of actinomycetes in microbial cave populations (Groth and SaizJimenez 1999; Groth et al. 1999, 2001). The type of Actinobacteria was most frequently isolated from bacterial consortia of Pajsarjeva jama (Pasic et al. 2010), Wind Cave (Chelius and Moore 2004), Altamira and Tito Bustillo caves (Schabereiter-Gurtner et al. 2002a, 2002b). In lava caves, Actinobacteria OTUs were one of the most numerous among all identified OTUs (Hathaway et al. 2014; Northup et al. 2011). Organic and inorganic products of their metabolism were reported to have a destructive effect on rock paintings present in these caves (Schabereiter-Gurtner et al. 2004). Evaluation of the species composition using nonculturing techniques showed that Actinobacteria formed a dominant part of the microbial community (80% of the population) in the Carlsbad Cavern (Barton et al. 2007) on rocks with CaCO3. Pseudonocardia spp. constitute half of the actinomycetes that inhabit this cave. Pseudonocardia are aerobes metabolizing a wide range of complex organic compounds. In Pajsarjeva jama, Actinobacteria were the second largest taxonomic type (16.3% of the sequences) (Pasic et al. 2010), as it was the case in extremely acidic environment (pH 0–1) in the Grotta del Fiume of the Frasassi cave system. The snottite material from the rocks of the Grotta del Fiume contained Actinobacteria represented only by the type Acidimicrobium. These bacteria are heterotrophs oxidizing sulfur, often encountered in extremely acidic environments. Actinobacteria formed the second most numerous group of microorganisms in the Llonin Cave colonizing the rock (22.2% of 16S rRNA sequences) (Schabereiter-Gurtner et al. 2004), while in Tito Bustillo and La Garma caves they were the third most numerous taxonomic type (9.8% and 19%, respectively) (Schabereiter-Gurtner et al. 2002b; 2004). In the white aggregations from the Altamira Cave, actinomycetes were one of the largest groups of bacteria (41.9% of 16S rRNA sequences from total DNA). They were represented by genera Pseudonocardia, Propionibacterium, Corynebacterium, Gordonia and


Microbial Diversity in Caves Frankia. In the remaining colonies they were sparse, but metabolically active. Actinobacteria was the second largest group in the Kartchner Cave (Ikner et al. 2007), regardless of the intensity of tourism, and in the Cave of Bats (41.3% of the microbial population). Phototrophic biofilm from the Cave of Bats contained Arthrobacter, Curtobacterium, Promicromonospora, Rhodococcus, Nocardia, Micrococcus, Blastococcus, Streptomyces and Kribbella genera of actinomycetes. The temperature in the cave varies depending on the location, from 8 to 14 C, humidity is 54 to 94%. Similarly, in two pools from the vadose zone in the Barenschacht Cave Actinobacteria are the second and third most numerous group (Shabarova and Pernthaler 2010). The cultivable representatives of this group were also isolated from crystals and walls of the Cave of Crystals in Chihuahua Mexico, which is characterized by constant temperature of 55 C and a humidity of 100%. All isolates were closely related to the genus Prauserella belonging to the family Pseudonocardiaceae (Quintana et al. 2013). Several representatives of Actinobacteria can be found in other caves, listed in Table 1. Acidobacteria The group of Acidobacteria is encountered in various terrestrial ecosystems, also in caves and acidic mines (Baker and Banfield 2003; Brofft et al. 2002). Genetic and metabolic diversity of this group may be comparable to the highly diverse Proteobacteria (Hugenholtz et al. 1998; Quaiser et al. 2003). In the beginning, microorganisms of this type were divided into 8 subgroups (Hugenholtz et al. 1998), but studies conducted by Barns et al. (2007) increased the number of subgroups to 26. In the study by Zimmermann and colleagues (2005a), aimed solely at understanding the species composition of Acidobacteria in the Altamira Cave, a preliminary analysis showed that 25% of the microorganisms identified belonged to subgroups 3–7 and 9–11. This type is an ecologically important member of the microbial population not only in the Altamira Cave (Schabereiter-Gurtner et al. 2002a), but also in the caves of Tito Bustillo (Schabereiter-Gurtner et al. 2002b) and La Garma (Schabereiter-Gurtner et al. 2004) as well as in the soil of the Niu Cave (Zhou et al. 2007), Wind Cave sediments (Chelius and Moore 2004), lava caves (one of the most numerous group) (Hathaway et al. 2014; Northup et al. 2011) and biofilm in the Roman catacombs (Zimmermann et al. 2005b). In other studies on the species composition of the caves, microorganisms belonging to this type were fairly common, although in most cases they did not constitute the dominant group, e.g., in the Pajsarjeva jama, they represented only 10.5% of the population, which was composed solely of subgroups 3 and 4. In the Lower Kane Cave, containing large amounts of sulfur, acidobacterial population was dominated by subgroup 7 and 8, and additionally contained a low number of subgroup 6 (Meisinger et al. 2007). These microorganisms were likely involved in this cave in the dissimilatory reduction of iron ions. By applying the FISH method it was documented that Acidobacteria

11 constituted from 5 to 10% of all microorganisms of the domain Bacteria in this environment. Engel et al. (2004) found that water mats of this group of bacteria were preferentially localized in oxygenated environments with low sulfide content. The study of Meisinger et al. (2007) has found that Acidobacteria mats always occurred near the dominant microorganisms belonging to the group of e- and/or g-Proteobacteria, which were likely chemolithoautotrophs (Engel et al. 2003, 2004). These observations may suggest that Acidobacteria are chemoorganotrophs growing near autotrophs, in environments poor in nutrients. These organisms comprised a small group in the Grotta Sulfurea of the Frasassi cave system (Macalady et al. 2006), Herrenberg Cave (Rusznyak et al. 2012), Carter Saltpeter Cave (Carmichael et al. 2013), Weebubbie Cave (Tetu et al. 2013) and also in the Barenschacht Cave (Shabarova and Pernthaler 2010). Although recent studies, carried out using culturing techniques, allowed researchers to detect representatives of subgroups 1, 2, 3, 4 and 8 (Coates et al. 1999; Janssen et al. 2002; Joseph et al. 2003; Kishimoto et al. 1991; Liesack et al. 1994; Sait et al. 2002, 2006; Stevenson et al. 2004), most Acidobacteria identified by sequence analysis of 16S rRNA remains uncharacterized and impossible to culture. Bacteroidetes Microorganisms of this type are phenotypically diverse, aerobic or facultatively anaerobic chemoorganotrophs, often producing carotenoids and/or flexirubin which confer yellow or orange colony coloration. This group contains psychrophilic, mesophilic and thermophilic bacteria. Although Bacteroidetes microorganisms are often found in caves, knowledge about their functional role in these ecosystems is limited. It is suggested that they are involved in the fermentation process and circulation of metal elements (Angert et al. 1998; Chelius and Moore 2004; Ikner et al. 2007; Macalady et al. 2006). This phylum constituted the largest group in biofilms developing on ferromanganese deposits of the Carter Saltpeter Cave (Carmichael et al. 2013). Cultivable isolates Flavobacterium sp. E8 and Flavobacterium sp. MTFA were actively engaged in formation of ferromanganese deposits by their capacity to oxidize Mn(II). Bacteroidetes were the second largest group of microorganisms in the white colonies inhabiting the rocks of the Altamira Cave. This type was represented by only a few metabolically active bacteria, mostly of the genus Flavobacterium (Portillo et al. 2009). In the Llonin Cave, Bacteroidetes were the third most dominant group (11.1% of 16S rRNA sequences) (Schabereiter-Gurtner et al. 2004). In the sediments of the Herrenberg Cave, Bacteoridetes were represented most frequently in identified16S rRNA sequences, constituting about 20% of all sequences. Several representatives of Bacteoridetes could be found in the following caves: Grotta Nuova di Rio Garrafo (Jones et al. 2010), Movile (Chen et al. 2009), Pajsarjeva jama (Pasic et al. 2010), Wind Cave (Chelius and Moore 2004), Grotta Sulfurea Frasassi cave system (Macalady et al. 2006), Lower Kane (Engel et al. 2004; Engel et al. 2010), La Garma (Schabereiter-Gurtner et al. 2004), Niu (Zhou et al. 2007), Tito

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Bustillo (Schabereiter-Gurtner et al. 2002b), Karthner (Ikner et al. 2007), Carlsbad Cavern (Barton et al. 2007), Magura Cave (Tomova et al. 2012), lava caves (Hathaway et al. 2014; Northup et al. 2011), Weebubbie (Tetu et al. 2013), Barenschacht (Shabarova and Pernthaler 2010) and in the bacterial populations that colonize stalactites in the Herrenberg Cave (Rusznyak et al. 2012).

Firmicutes Microorganisms belonging to Firmicutes are often found in other ecosystems than caves and they are characterized by heterotrophic and chemolithoautotrophic growth. A few representatives are anaerobic photoheterotrophs. They can be aerobic, facultative anaerobic or strictly anaerobic bacteria and usually grow in neutral pH. Certain bacteria of this type colonize environments with acid or alkaline pH as well as sites with high temperature and salinity. Firmicutes are most often found in microbial populations inhabiting the surface of rock walls and sediments in caves. Some of these microorganisms are able to reduce or oxidize sulfur, e.g., Desulfotomaculum spp. and Sulfobacillus acidophilus (Macalady et al. 2007). They have a wide metabolic range that allows growth on various organic compounds. They also possess better resistance to stress caused by low amount of nutrients and dehydration when compared to Proteobacteria. This group dominated in the Kartchner Caverns, in areas of medium and low tourist traffic (52% and 66% of cultured isolates, respectively) (Ikner et al. 2007). Similarly, Firmicutes dominated in the phototrophic biofilm formed in the Cave of Bats (49.5% of 16S rRNA sequences) with medium tourist intensity (20,000 visitors per year) (Urzì et al. 2010). Microorganisms identified in this biofilm belonged to the genera: Bacillus, Peanibacillus and Staphylococcus. In the Lechuguilla Cave, Firmicutes were the largest group (39%), represented almost exclusively by the family Lactobacillaceae. These are anaerobic or microaerophilic chemoorganotrophs that utilize carbohydrate fermentation as an energy source. Most of the 16S rRNA sequences belonged to the genus Leuconostoc, which requires the presence of Mn2C ions for growth. The other two sequences belonged to Streptococcus sanguis, which is a common human pathogen, occurring in the cave as a result of contamination (Northup et al. 2003). In an environment of extremely acidic pH (0–1) in the Grotta del Fiume in Frasassi cave system, one representative of the Sulfobacillus genus has been identified, i.e., sulfur-oxidizing S. acidophilus, capable of auto-, mixo- and hetero-trophic growth (Norris et al. 1996). Firmicutes were poorly represented in the following caves: Movile (Chen et al. 2009), Herrenberg Cave (Rusznyak et al. 2012) Lower Kane (Engel et al. 2010), Lolin, La Garma (Schabereiter-Gurtner et al. 2004), Altamira (SchabereiterGurtner et al. 2002a), Magura (Tomova et al. 2012), lava caves (Hathaway et al. 2014; Northup et al. 2011), Weebubbie (Tetu et al. 2013) and Barenschacht (Shabarova and Pernthaler 2010).

_ and Zielenkiewicz Tomczyk-Zak Verrucomicrobia Verrucomicrobia represents a distinct phylogenetic lineage of ubiquitous bacteria with a small number of cultured species. Cells of some possess cellular extensions and intracellular compartments.They can be found in a variety of habitats, such as soils and fresh and marine waters, human intestines as well as very extreme environments, including hot springs and Antarctica. They are one of the most abundant bacterial groups present in soils and marine waters (Bergmann et al. 2011; Op den Camp et al. 2009). Members of this phylum are strict aerobes, facultative anaerobes or strict anaerobes.Some are autotrophs that grow by oxidizing methane at high temperature in strongly acidic environments (Freitas et al. 2012). Bacteria belonging to the type Verrucomicrobia are mostly chemoheterotrophs assigned to 7 taxonomic classes, showing preference to certain environments. This type was represented by a small number of microorganisms in Movile (Chen et al. 2009), Herrenberg Cave (Rusznyak et al. 2012), Grotta Sulfurea Frasassi cave system (Macalady et al. 2006), Lower Kane (Engel et al. 2010), Barenschacht (Shabarova and Pernthaler 2010), Wind Cave (Chelius and Moore 2004), Altamira (Portillo et al. 2009), Carter Saltpeter Cave (Carmichael et al. 2013), lava caves (Hathaway et al. 2014; Northup et al. 2011) and Pajsarjeva jama (Pasic et al. 2010). The ecological role of Verrucomicrobia in cave ecosystems is not recognized.

Planctomycetes It is a cosmopolitan group of microorganisms inhabiting a wide variety of environments, highly abundant in acidic wetlands and marine detritus (Fuchsman et al. 2012; Ivanova and Dedysh 2012). The characteristic features of these microorganisms include the lack of murein in proteinaceous cell wall and the presence of unique intracellular compartments. The majority of Planctomycetes are aerobic or facultatively anaerobic chemoheterotrophs. The separate anammox bacteria are primarily autotrophic, able to carry out anaerobic ammonia oxidation coupled with nitrate reduction (Fuerst and Sagulenko 2011). Planctomycetacia degrading complex organic matter under both aerobic and anaerobic conditions play an important role in global carbon and nitrogen cycles. These bacteria are often found in caves, but their ecological role in these ecosystems is not known. Planctomycetes composed numerous group in the soil of the Niu Cave (9% of 16S rRNA sequences - the third most numerous group) (Zhou et al. 2007) and in the white colonies of the Altamira Cave (18.8% of 16S rRNA sequences - the second most numerous group) (Portillo et al. 2009). Planctomycetes were poorly represented in the following caves: Movile (Chen et al. 2009), Herrenberg (Rusznyak et al. 2012), Pajsarjeva jama (Pasic et al. 2010), Wind (Chelius and Moore 2004), the Grotta Sulfurea Frasassi cave system (Macalady et al. 2006), Lower Kane (Engel et al. 2010), Barenschacht (Shabarova and Pernthaler 2010), Carter Saltpeter (Carmichael et al. 2013), lava caves (Northup et al.



2011, Hathaway et al. 2014), Weebubbie (Tetu et al. 2013) and Tito Bustillo (Schabereiter-Gurtner et al. 2002b). Chloroflexi The Chloroflexi phylum, formerly known as “green nonsulfur bacteria,” is a systematic group with a small number of cultivated representatives, but with rapidly accumulating sequences identified in metagenomic studies. They are found in various natural and engineered environments, such as sediments, soils, rivers, oceans and granular sludges, including hot and cold niches, where they occasionally constitute dominating taxons (Yamada and Sekiguchi 2009). This type is represented by physiologically diverse aerobic or facultative aerobic chemoheterotrophs, obligatory or facultative phototrophs and obligately anaerobic dehalorespiring bacteria. They are morphologically diverse but prevalently filamentous. Many of them establish syntrophic relations, especially in granules formed with methanogens. They seem to be indispensable in waste and wastewater treatment systems. Such bacteria may also play an important role in bioremediation of contaminated soils and groundwaters. Microorganisms of this group comprise usually a small fraction of the population. Engel et al. (2010) were the only ones who identified noncultured microorganisms of the type Chloroflexi as one of the dominant groups (14% of the sequences) in the gray water mats of the Lower Kane Cave, which have anaerobic conditions. Their ecological role in the cave ecosystem has not been determined. Several microorganisms of this type have been identified in the following caves: Grotta Nuova di Rio Garrafo (Jones et al. 2010), Pajsarjeva jama (Pasic et al. 2010), Niu (Zhou et al. 2007), Tito Bustillo (Schabereiter-Gurtner et al. 2002b), La Garma (Schabereiter-Gurtner et al. 2004), Altamira (Portillo et al. 2009; Schabereiter-Gurtner et al. 2002a), lava caves (Hathaway et al. 2014; Northup et al. 2011), Weebubbie (Tetu et al. 2013) and in fluvial sediments of the Herrenberg Cave (Rusznyak et al. 2012).

Groups of Bacteria Rarely Found in Caves The following taxonomic groups of bacteria are rarely identified in the studies on the biodiversity of caves. Reasons for their infrequent occurrence in these ecosystems may be associated with specific selection factors of these environments, or the fact that they are few in number and thus they are not always detected. The latter reason is associated with such critical steps in the evaluation of biodiversity as imperfect isolation of total DNA or suboptimal amplification reaction of a molecular marker. Another reason may be that there are no representatives of these microorganisms in the databases, so such sequences would be considered unclassified. Not without significance is the choice of research method for biodiversity studies. The use of next generation whole metagenome sequencing of environmental samples could reveal new or not numerous genera and lead to identification of bacterial groups rarely found in caves.

13 Gemmatimonadetes Microorganisms of this type are mesophilic heterotrophs growing in aerobic conditions, found in many terrestrial and aquatic environments. This group is rarely encountered in cave ecosystems and its ecological role in these environments is not known. Representatives of the Gemmatimonadetes type have been identified in the microbial population colonizing walls of the Pajsarjeva jama (Pasic et al. 2010), Herrenberg Cave fluvial sediments (Rusznyak et al. 2012), soil of Niu Cave (Zhou et al. 2007), yellow and white mats of lava caves (Hathaway et al. 2014; Northup et al. 2011) and yellow colonies in the Altamira Cave (Portillo et al. 2008). In all of these environments, Gemmatimonadetes represented a small percentage of the population. Databases containing environmental sequences indicated that microorganisms belonging to this type are widely distributed in nature, and their 16S rRNA sequences have a divergence of 19%, which is higher than the well-known divergence of actinomycetes (18%) (Zhang et al. 2003). Spirochetes Microorganisms of Spirochetes type are chemoorganotrophs, growing as aerobic, microaerophilic, facultatively anaerobic or strictly anaerobic organisms. This type is very rare in cave ecosystems, and was represented by a small number of bacteria in the water mats of Grotta Nuova di Rio Garrafo (Jones et al. 2010), Movile (Chen et al. 2009), Lower Kane (Engel et al. 2010) and Wind Cave (Chelius and Moore 2004) caves. Ecological role of Spirochetes in cave ecosystems is not known. Cyanobacteria Cyanobacteria are aerobic microorganisms developing only in the presence of natural or artificial light, hence they are only found in illuminated parts of caves. All known bacteria of this type are capable of photoautotrophy, although chemoheterotrophs are also encountered. Numerous Cyanobacteria have been identified in phototrophic biofilm growing on the rocks at the entrance and exit of the Cave of Bats (Urzì et al. 2010). These bacteria belong to the orders Chroococcales and Nostocales. Chemoorganotrophic bacteria often grow in the vicinity of these microorganisms, in a common fibrillar matrix. A large variety of chemoorganotrophs was correlated with the high biodiversity of Cyanobacteria. Artificial light installed for tourists in the cave of Tito Bustillo induced strong growth and colonization of stalagmites and stalactites by Scytonema julianum, Geitleria calcarea and Pseudocapsa sp. (Schabereiter-Gurtner et al. 2002b). These microorganisms were also found in lava caves (Hathaway et al. 2014; Northup et al. 2011) and slimes of Weebubbie cave (Tetu et al. 2013). Chlorobi Microorganisms of this type are complete anaerobes, obligatorily phototrophic, able to utilize thiosulfates, and sulfide as electron donors for the assimilation of CO2. They are usually found in aquatic environments. It is a type very rarely found in caves, identified only in small numbers in water mats of the Frasassi cave system at pH » 7.3 (Macalady et al. 2006),

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slimes of Weebubbie cave (Tetu et al. 2013) and in ferromanganese deposits of the Carter Saltpeter Cave (Carmichael et al. 2013). Fibrobacteres This type is also rarely found in caves, identified only in small numbers in the epiphreatic zone pool of the Barenschacht Cave (Shabarova and Pernthaler 2010), and in the bacterial population of the Herrenberg Cave stalactites (Rusznyak et al. 2012). The ecological role in these ecosystems is not known. Little is known as well about the metabolism of this group of bacteria. Thanks to the development of research methods, the fast-growing resources of databases and improved bioinformatic analysis tools, new or very rare organisms are increasingly being discovered. For example, in the Grotta Nuova di Rio Garrafo, in addition to the previously described taxonomic groups, representatives of Elusimicrobia, MVP-5, OP 5 and OP 11 have been also identified (Jones et al. 2010). Notwithstanding, singular cases of identified taxonomic groups have been omitted in this review if their role in the environment has not been characterized.

Archaea The unique distinguishing traits of morphology and physiology of certain single-cell organisms led to establishing a separate domain of life- the Archaea. For a very long time Archaea organisms were attributed solely to extreme environments (Woese et al. 1990). However, subsequent studies demonstrated the presence of Archaea microorganisms in soil samples (DeLong 1998), in caves and other ecosystems. Thus, archaeons are no longer considered extremophilic organisms. In this domain, the identified microorganisms can be aerobic, facultatively or strictly anaerobic, chemolithotrophic, chemoheterotrophic, acidophilic, thermophilic or halophilic. Most of these organisms are able to metabolize S0. They are the only organisms known to carry out methanogenesis processes in syntrophy with bacterial hydrogen producers in anoxic conditions. Archaeons are also able to carry out anaerobic methane oxidation as well as aerobic ammonia oxidation (Cavicchioli 2011). Due to these properties, archeons play an important part in the global carbon and nitrogen cycles. Up to today, Crenarchaeota and Euryarchaeota are the most distinguished groups within the Archaea domain, but rapidly growing new archeal sequences lead to proposing more subdivisions. No pathogens have been found in this domain so far. Little is known about the occurrence of Archaea in cave systems. Frequently identified types of archaeans belong to Crenarcheaota and Euryarcheaota. Sequences of 16S rRNA of both types were found, e.g., in the 16S rRNA gene library of water mats from the Movile Cave (Chen et al. 2009) and sediments of the Wind Cave (20 sequences of Euryarcheaota and 2 of Crenarcheaota) (Chelius and Moore 2004). In another cave with low pH (pH 0–1; Grotta del Fiume) that

Fig. 3. Ideogram shows influence of the environmental physicochemical factors on microbial diversity in caves. The size of the opening angle of the diamond is inversely proportional and the intensity of the color directly proportional to the degree of biodiversity.

belongs to the Frasassi cave system, a group of Archaea microorganisms was identified and determined to account for 40% of the microorganisms identified in the snottites (Macalady et al. 2007). No representatives of Crenarcheaota were found in this cave, and the population was dominated mainly by the genus Ferroplasma, which is able to oxidize reduced sulfur compounds (Okibe et al. 2003). Microorganisms of the genus Ferroplasma are autotrophs (Golyshina et al. 2000), which are able to adapt to the heterotrophic or mixotrophic metabolism, depending on the environment (Tyson et al. 2004). Studies carried out in acidic environments showed a correlation between pH and the domination of Ferroplasma (Golyshina and Timmis 2005). Additionally, Thermoplasma organisms were also found in this population and other archaeans, which were phylogenetically related to archaeans from acid mine waters and hot water streams in Yellowstone. In recent years in the Altamira Cave, a new branch of psychrophilic Crenarcheota microorganisms have been identified. They are metabolic components of yellow, gray and white colonies covering the walls of this cave (Gonzales et al. 2006). Role of these microorganisms in the environment remains unclear. Schleper et al. (2005) have suggested that low-temperature Crenarcheota may be capable of oxidizing ammonium. Mesophilic Crenarcheota and Euryarcheota have been identified in the Lechuguilla Cave (51 sequences of 16S rRNA belonging to Crenarcheota, 5 sequences to Euryarcheota; Northup et al. 2003). Recently, Tetu et al. (2013) identified in slimes of Weebubbie cave an abundant population of Thaumarchaeota (»45% of all sequences) that was related to the ammonia oxidizing Nitrosopumilus maritimus. Few Archaea were identified in ferromanganese deposits of the Carter Saltpeter Cave (Carmichael et al. 2013), water mats of Grotta Sulfurea from the Frasassi cave system (pH » 7.3) (Macalady et al. 2006), as well as in water mats of the Lower Kane Cave (only Euryarcheota represented by Archaeoglobus spp., which reduces sulfur) (Engel et al. 2010).



Conclusions The caves that are separated and at the same time remain open ecosystems, with stable physical and chemical conditions, allow evaluating the structure of the microbial community in respect to specific factors. They also enable studying relationships between particular organisms in the natural environment, and to track changes dependent on small fluctuations. Numerous studies undertaken in the last decade demonstrated abundant and diverse biota in cave ecosystems. Out of all organisms inhabiting caves, prokaryotes have major effect on these environments. Independently of their geological origin, caves undergo permanent changes, which are influenced by the activity of organisms living there. Especially microbial biofilms, developing on cave walls, sediments or waters, due to their abundance and specific community physiology, affect the state of these niches. Biogeochemical processes are becoming a well-recognized and documented field (Gadd 2010). Examination of these processes allow determining the ability of microbes to transform minerals, change the air composition or water pH, all of which can lead to reshaping the cave. The knowledge of individual microbes as well as entire communities from diverse caves together with their physiological potential has already found practical applications in variety of disciplines. Spectacular development of ideas involving microbes with adequate physiological properties can be observed especially in biomining and bioremediation technologies. At the same time understanding of biogeological processes is very important for designing preservation strategies to maintain paleolitical cave paintings and other historical monuments. As shown by systematic monitoring of several historically important caves (Lascaux Cave, Altamira Cave) affected by human activities, the use of chemical preservations in biocide treatment led to promotion of secondary colonization, including bacterial and fungal human pathogens (Bastian et al. 2010, Saiz-Jimenez et al. 2011). It seems that the best way to prevent deterioration of caves is monitoring the microbial diversity and influx of organic matter level by establishing a surveillance system of environmental conditions and limiting of human access. We can also educate visitors on how to keep from enriching caves with organic matter, which can limit some of the human impact. Taxon distribution in caves characterized by extreme, diverse physico-chemical conditions, presented in this work indicates high microbial biodiversity and at the same time preferential occupancy of caves by particular taxonomic groups. Bacterial classes, such as a-, b- or g-Proteobacteria are metabolically diverse enough to be able to exist in many different environments. The most frequently found microorganisms in caves are g-Proteobacteria, which were detected in almost all types of caves, including sulfur caves, irrespectively of the examined substrate. Such dominance may be associated with the fact that this group of bacteria contains many chemotrophic representatives which cope well in oligotrophic environments. Heterotrophic actinobacteria were also frequently noted. Their presence could be observed in every examined cave, excluding three caves characterized by high concentration of

15 sulfur compounds. Their prevalence may be also related to their diverse metabolism and ability to use as a source of energy organic matter produced by commonly present chemolithotrophs. In turn, e-Proteobacteria are an example of microorganisms preferentially residing in sulfur-rich environments, which is their main source of energy. However, the increasing repertoire of methods applied to analyze specific environmental niches, especially the next generation sequencing, expands the image of biodiversity practically in every examined environment. Determination of prokaryotic diversity in caves described above, in spite of numerous studies, is far from complete, for several reasons: 1. Not all accessible caves have been explored. Thousands of caves and cave systems on Earth are known and new are still discovered, but only a small number has been studied so far. In general, explored caves exhibit some special features, such as unusual geochemical or biological structures, presence of ancient paintings, archaeological importance, extreme conditions, etc. Vast number of “less interesting” caves remain unexplored. 2. Inconsistency in presented data concerning studied caves, including both physical and/or chemical data, and the preference to examine particular groups of organisms. There is no obligatory format in presenting data; particularly inconsistent is the range of physico-chemical parameters examined. Moreover, while some scientific works concentrate only on the overall biodiversity, others deal with a particular group, e.g., cultivated organisms or microbes exhibiting special metabolic activity. Detailed description of physicochemical parameters would provide information critical for understanding the presence of a particular group of microorganisms in given niche. 3. Differences in applied methodology and statistical analysis. This is the most important factor. Different methods used to discover and statistics applied to analyze biodiversity make decent comparisons between cave ecosystems difficult. It was shown in many studies that: older popular techniques (as DGGE or FISH) underestimate diversity due to limitations of visual resolution, all currently used PCR-based techniques, including pyrotags, have a permanent ingenious bias, while 16S rRNA sequencing strongly depends on the choice and specificity of used primers. Deep sequencing of metagenomes provide probably the largest data but still should be supported by others strategies especially cultivation methods (Donachie et al. 2007). A separate yet also important problem is insufficient sampling. Even in different places of the same cave biodiversity could be different as can be inferred from already mentioned Ortiz et al. (2013). They analyzed the biodiversity of speleothems in the Kartchner Caverns and showed that in spite of stable conditions in the cave and close proximity between the studied objects, biodiversity was quite different in one speleothem probably due to higher metal content in this place. 4. Dynamic natural changes in diversity caused by external factors. Biodiversity can alter due to sporadic singular events: geological, climatic or biological. For example, earthquakes can cause collapse of the entrance, leading to


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changes of atmosphere in the cave. Flooding can be a source of external matter, causing prevalence of heterotrophic organisms or physically disturb/damage the biofilm. Changes in organic matter and appearance of new organisms in caves can be also connected with the presence of animals or humans. Moreover, special attention should be given to the effects of nematode predation on biofilms (Jousset 2012). Cave ecosystems described previously in literature seem to confirm that, as generally accepted, diversity of the microbial environment is positively correlated with the increase of its heterogeneity. As shown in Figure 3, intensification of a particular physical or chemical factor, and the availability of specific substances, which are a source of necessary nutrients, determines the structure of microbial communities in a particular niche and the development of specific physiological interrelationships. Consequently, in ecosystems where particular factor(s) are extreme, the diversity decreases as manifested, e.g. for extremely acidic (Macalady et al. 2007) or hot (Quintana et al. 2013) caves. Such biodiversity “pattern” in caves of generally similar physical-chemical conditions has been recently described in excellent reviews (Banerjee and Joshi 2013; Lee et al. 2012; Romero 2012). The presented picture of microbial life in caves does not exhaust all existing niches with their often peculiar community compositions and interrelations or roles of specific individual organisms. Employment of fast and cost-effective high throughput sequencing technologies should enable deeper insight into the diversity of a multitude of yet unexplored environments. To draw a complete picture of the relationships between microorganisms colonizing these ecosystems, it seems necessary to standardize the means of describing environmental conditions, agree on the common methodology and statistical evaluation of results, which would be obtained from much more extended sampling.

Acknowledgments We acknowledge prof. J. Bardowski for critical reading of the manuscript.

Funding This article was supported by strategic research project No. SP/J/3/143045/11 from The National Center for Research and Development (NCBiR), Poland over the period 2011– 2014.

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