**3. Results and discussion**

Cavernicolous cyanobacteria belong to the orders Chroococcales, Nostocales, Oscillatoriales, and Stigonematales. Chroococcales are the most common order represented by genera: *Aphanocapsa, Aphanothece, Chondrocystis, Chroococcidiopsis, Chroococcus, Gloeocapsa, Gloeocapsopsis,* and *Gloeothece.* Whereas among Nostocales were present only three genera: *Hassalina, Nostoc,* and *Scytonema.* Coccoid and filamentous cyanobacteria species were dominant in the entrance zone, while in the dim light zone they were encountered only sporadically. Czerwik-Marcinkowska and Mrozińska [20] and Martínez and Asencio [21] stated that coccoid forms are more abundant in dark areas, whereas filamentous forms tend to be more diverse in illuminated part of cave, unlike the findings of Vinogradova et al. [22]. Epilithic cyanobacteria are the first colonizers and play an important role in the genesis of biofilms, being able to produce exopolymeric substances (EPS) that allow the adhesion to rocks and the consequent establishment of a microbial community [7]. Besides the colonization of various stone substrata and the production of pigments that are responsible for colored effects on rocky cave walls and erosion of the stone substrata, they can also serve as a food source for animals. Almost all cavernicolous cyanobacteria have gelatinous extracellular sheath layers of various thickness composed of polysaccharides. Keshari and Adhikary [23] observed that the gelatinous extracellular sheath of cyanobacteria plays a crucial role in adhesion to the substratum and also acts as a water reservoir, thus enabling the cyanobacteria to survive drought periods. Pattanaik et al. [24] suggested that water stress proteins, glycan, and UVA/B absorbing pigments are the main components of the EPS of cyanobacteria. The genus *Gloeocapsa* has the most various colorations due to the presence of a pigment called gloeocapsin. Another wellstudied pigment, scytonemin, causes the dark coloration of cyanobacterial crusts [24]. Genera that usually dominate dark-colored crusts are *Scytonema, Nostoc,* and *Tolypothrix.* Some cyanobacteria from genus *Scytonema* have calcified trichomes [25]. In **Table 1**, the most frequent and abundant cyanobacteria species found in different European caves based on literature results since 2010 are reported.

**Cyanobacteria Origin References** *Aphanocapsa fusco lutea* Božana Cave (Serbia) Popović et al. [28] *Aphanocapsa muscicola* Božana Cave (Serbia) Popovć et al. [28]

*Asterocapsa divina* Gelda Cave (Spain) Martínez & Asencio [21]

Ojców National Park (Poland)

*Chroococcus westii* Gelda Cave (Spain) Martínez & Asencio [21] *Cyanobacterium cedrorum* Gelda Cave (Spain) Martínez & Asencio [21] *Cyanosaccus aegeus* Gelda Cave (Spain) Martínez & Asencio [21] *Cyanosaccus atticus* Gelda Cave (Spain) Martínez & Asencio [21] *Cyanostylon microcystoides* Gelda Cave (Spain) Martínez & Asencio [21] *Gloeocapsa atrata* Božana Cave (Serbia) Popović et al. [28]

(Serbia), caves from Ojców National

*Chondrocystis dermochroa* Božana Cave (Serbia) Popović et al. [28] *Chroococcidiopsis kashayi* Božana Cave (Serbia) Popović et al. [28]

*Chroococcus pallidus* Božana Cave (Serbia) Popović et al. [28] *Chroococcus spelaeus* Gelda Cave (Spain) Martínez & Asencio [21]

Martínez & Asencio [21], Popović et al. [28]

Diversity of Cyanobacteria on Limestone Caves http://dx.doi.org/10.5772/intechopen.79750 49

Popović et al. [28], Czerwik-Marcinkowska

Martínez & Asencio [21], Popović et al. [28], Czerwik-Marcinkowska et al. [26]

Popović et al. [28], Czerwik-Marcinkowska

Martínez & Asencio (2010), Popović et al. [28], Czerwik-Marcinkowska et al. [26]

Popović et al. [28], Czerwik-Marcinkowska

Czerwik-Marcinkowska et al. [26]

Czerwik-Marcinkowska et al. [26]

et al. [26]

et al. [26]

et al. [26]

*Aphanothece saxicola* Gelda Cave (Spain), Božana Cave (Serbia)

*Calothrix fusca* Caves from Ojców National Park (Poland)

*Chroococcus ercegovicii* Božana Cave (Serbia), caves from

*Chroococcus turgidus* Caves from Ojców National Park (Poland)

*Gloeocapsa biformis* Gelda Cave (Spain), Božana Cave

*Gloeocapsa punctata* Božana Cave (Serbia), caves from

*Gloeocapsa rupicola* Gelda Cave (Spain), Božnana Cave

*Gloeothece palea* Božana Cave (Serbia), caves from

Park (Poland)

*Gloeothece cyanochroa* Božana Cave (Serbia) Popović et al. [28]

*Gloeocapsopsis dvorakii* Božana Cave (Serbia) Popović et al. [28]

Park (Poland)

*Gloeocapsa compacta* Božana Cave (Serbia) Popović et al. [28] *Gloeocapsa lignicola* Božana Cave (Serbia) Popović et al. [28] *Gloeocapsa nigrescens* Gelda Cave (Spain) Martínez & Asencio [21] *Gloeocapsa novacekii* Gelda Cave (Spain) Martínez & Asencio [21]

Ojców National Park (Poland)

Ojców National Park (Poland)

(Serbia), caves from Ojców National

#### **3.1. Ecology of cyanobacteria in caves**

Whitton [25] stated that cyanobacteria have existed for 3.5 billion years and they are the most important photosynthetic organisms on the planet for cycling carbon and nitrogen. Cyanobacteria living in limestone caves present a unique group of microorganisms, which developed adaptations against the more or less extreme conditions of their habitats. They play an important role in several aspects of the environment such as colonizers, nitrogen fixers, prey for micrograzers, or deterioration agents. Cyanobacteria are morphologically diverse group of prokaryotes successfully colonizing and inhabiting almost all kind of terrestrial and aquatic habitats including extreme microhabitats such as caves, rocks, external walls of monuments, and buildings [26, 27]. Wild caves and caves made accessible to general public are characterized by extreme conditions, and they also offer a unique habitat for cyanobacteria [20]. Cyanobacteria are prone to environmental stress such as desiccation, temperature, and UV radiation, and they adopted survival strategies by producing


**3. Results and discussion**

48 Cyanobacteria

since 2010 are reported.

**3.1. Ecology of cyanobacteria in caves**

Cavernicolous cyanobacteria belong to the orders Chroococcales, Nostocales, Oscillatoriales, and Stigonematales. Chroococcales are the most common order represented by genera: *Aphanocapsa, Aphanothece, Chondrocystis, Chroococcidiopsis, Chroococcus, Gloeocapsa, Gloeocapsopsis,* and *Gloeothece.* Whereas among Nostocales were present only three genera: *Hassalina, Nostoc,* and *Scytonema.* Coccoid and filamentous cyanobacteria species were dominant in the entrance zone, while in the dim light zone they were encountered only sporadically. Czerwik-Marcinkowska and Mrozińska [20] and Martínez and Asencio [21] stated that coccoid forms are more abundant in dark areas, whereas filamentous forms tend to be more diverse in illuminated part of cave, unlike the findings of Vinogradova et al. [22]. Epilithic cyanobacteria are the first colonizers and play an important role in the genesis of biofilms, being able to produce exopolymeric substances (EPS) that allow the adhesion to rocks and the consequent establishment of a microbial community [7]. Besides the colonization of various stone substrata and the production of pigments that are responsible for colored effects on rocky cave walls and erosion of the stone substrata, they can also serve as a food source for animals. Almost all cavernicolous cyanobacteria have gelatinous extracellular sheath layers of various thickness composed of polysaccharides. Keshari and Adhikary [23] observed that the gelatinous extracellular sheath of cyanobacteria plays a crucial role in adhesion to the substratum and also acts as a water reservoir, thus enabling the cyanobacteria to survive drought periods. Pattanaik et al. [24] suggested that water stress proteins, glycan, and UVA/B absorbing pigments are the main components of the EPS of cyanobacteria. The genus *Gloeocapsa* has the most various colorations due to the presence of a pigment called gloeocapsin. Another wellstudied pigment, scytonemin, causes the dark coloration of cyanobacterial crusts [24]. Genera that usually dominate dark-colored crusts are *Scytonema, Nostoc,* and *Tolypothrix.* Some cyanobacteria from genus *Scytonema* have calcified trichomes [25]. In **Table 1**, the most frequent and abundant cyanobacteria species found in different European caves based on literature results

Whitton [25] stated that cyanobacteria have existed for 3.5 billion years and they are the most important photosynthetic organisms on the planet for cycling carbon and nitrogen. Cyanobacteria living in limestone caves present a unique group of microorganisms, which developed adaptations against the more or less extreme conditions of their habitats. They play an important role in several aspects of the environment such as colonizers, nitrogen fixers, prey for micrograzers, or deterioration agents. Cyanobacteria are morphologically diverse group of prokaryotes successfully colonizing and inhabiting almost all kind of terrestrial and aquatic habitats including extreme microhabitats such as caves, rocks, external walls of monuments, and buildings [26, 27]. Wild caves and caves made accessible to general public are characterized by extreme conditions, and they also offer a unique habitat for cyanobacteria [20]. Cyanobacteria are prone to environmental stress such as desiccation, temperature, and UV radiation, and they adopted survival strategies by producing


envelopes and sheaths, tend to be dominant in terms of biomass. Cyanobacteria play a main role in the species biodiversity of caves. However, characterizing the biodiversity of caves is challenging because cyanobacterial communities often have high richness and contain numerous species that have neither been isolated nor described using traditional culturing techniques. The culture-independent methods can be applied to study cave communities and are especially powerful if combined with culture-based information. Many studies [20, 30] have described cyanobacteria occurring in both terrestrial sediments and aquatic cave environments around the world. This widespread distribution reflects the tolerance of cyanobacteria toward environmental stress due to a broad spectrum of specific properties in physiology [31]. Jones and Motyka [32] noted that a single microorganism can change from an autotroph (utilizing light for food) to a mixotroph (autotrophic microorganism that grows more rapidly in the presence of certain organic substrate) to a heterotroph (growth with no light). Most of these cavernicolous species are nonresidents transported into caves by water, air, sediment, and animals. Moreover, these enrichment-based and cultural studies have focused on typical heterotrophic microorganisms, which grow in an extreme environment [33]. Culture-independent, molecular phylogenetic techniques have since shown that many previously unknown species can be found in caves [34]. Impact of cave tourism (artificial light) is altering the natural light gradient in cave ecosystems, which may have important repercussions on the composition of cyanobacteria communities inside the caves, and that is why, lampenflora can be regarded as invasive [35]. Tourists entering limestone caves are responsible for transferring cyanobacteria spores [36], leading to unintentional biological pollution and favoring, at the same time, the colonization of other cave microorganisms [37]. As a consequence, the alteration of the natural environmental conditions in caves may also modify the cyanobacteria communities. Hobbs et al. [38] demonstrated that lampenflora does not grow at close distance from incandescent lights due to high temperature. However, the artificial illumination also influences the water content of the substrate and air. Tourist presence leads to the increase of both temperature and CO<sup>2</sup> concentration inside cave [15], intensifying wall erosion [39]. Cyanobacteria communities in caves are mainly composed of cavernicolous species, generally characterized by small size, resistance to desiccation, specific preferences for pH, and tolerating low nutrient levels and high conductivity. Saiz-Jimenez [40] showed that cave environment is in a constant battle to remove cyanobacteria and lint left by visitors without damaging underlying formations. Bright artificial light installed in caves for the benefits of visitors may adversely effect on drying out surfaces and decreasing relative humidity, which may be lethal to cave adapted microorganisms. Moist, humid conditions favor the growth of species on soils and rocks, for instance, *Nostoc commune* colonies which typically grow on calcareous soils or depressions on limestone surfaces. In limestone caves, cyanobacteria can be found in water bodies [41] and in subaerophytic karst habitats [42]. Cavernicolous cyanobacteria can be observed in the cave entrance illuminated by direct or indirect sunlight and caves equipped with artifi-

Diversity of Cyanobacteria on Limestone Caves http://dx.doi.org/10.5772/intechopen.79750 51

cial illumination, as a part of a lampenflora community around lamps [12].

Limestone caves are under immense pressure from anthropogenic activities, especially in recent years [9], and are probably one of the centers of biodiversity for certain types of cyanobacteria [2], especially for species from families Hapalosiphonaceae and Symphyonemataceae [43].

**Table 1.** Cyanobacteria species reported in caves based on literature results since 2010.

photoprotective pigments and bioactive compounds. Rock inhabiting cyanobacteria can be divided into "epilithic," colonizing the substrate surface directly; "hypolithic," living under pebbles and small stones lying on the rock; and "endolithic," living in an upper layer of rock [29]. The cavernicolous cyanobacteria species reported from rock and stone wall surfaces, mostly coccoid and heterocytous types with (often colored) mucilaginous envelopes and sheaths, tend to be dominant in terms of biomass. Cyanobacteria play a main role in the species biodiversity of caves. However, characterizing the biodiversity of caves is challenging because cyanobacterial communities often have high richness and contain numerous species that have neither been isolated nor described using traditional culturing techniques. The culture-independent methods can be applied to study cave communities and are especially powerful if combined with culture-based information. Many studies [20, 30] have described cyanobacteria occurring in both terrestrial sediments and aquatic cave environments around the world. This widespread distribution reflects the tolerance of cyanobacteria toward environmental stress due to a broad spectrum of specific properties in physiology [31]. Jones and Motyka [32] noted that a single microorganism can change from an autotroph (utilizing light for food) to a mixotroph (autotrophic microorganism that grows more rapidly in the presence of certain organic substrate) to a heterotroph (growth with no light). Most of these cavernicolous species are nonresidents transported into caves by water, air, sediment, and animals. Moreover, these enrichment-based and cultural studies have focused on typical heterotrophic microorganisms, which grow in an extreme environment [33]. Culture-independent, molecular phylogenetic techniques have since shown that many previously unknown species can be found in caves [34]. Impact of cave tourism (artificial light) is altering the natural light gradient in cave ecosystems, which may have important repercussions on the composition of cyanobacteria communities inside the caves, and that is why, lampenflora can be regarded as invasive [35]. Tourists entering limestone caves are responsible for transferring cyanobacteria spores [36], leading to unintentional biological pollution and favoring, at the same time, the colonization of other cave microorganisms [37]. As a consequence, the alteration of the natural environmental conditions in caves may also modify the cyanobacteria communities. Hobbs et al. [38] demonstrated that lampenflora does not grow at close distance from incandescent lights due to high temperature. However, the artificial illumination also influences the water content of the substrate and air. Tourist presence leads to the increase of both temperature and CO<sup>2</sup> concentration inside cave [15], intensifying wall erosion [39]. Cyanobacteria communities in caves are mainly composed of cavernicolous species, generally characterized by small size, resistance to desiccation, specific preferences for pH, and tolerating low nutrient levels and high conductivity. Saiz-Jimenez [40] showed that cave environment is in a constant battle to remove cyanobacteria and lint left by visitors without damaging underlying formations. Bright artificial light installed in caves for the benefits of visitors may adversely effect on drying out surfaces and decreasing relative humidity, which may be lethal to cave adapted microorganisms. Moist, humid conditions favor the growth of species on soils and rocks, for instance, *Nostoc commune* colonies which typically grow on calcareous soils or depressions on limestone surfaces. In limestone caves, cyanobacteria can be found in water bodies [41] and in subaerophytic karst habitats [42]. Cavernicolous cyanobacteria can be observed in the cave entrance illuminated by direct or indirect sunlight and caves equipped with artificial illumination, as a part of a lampenflora community around lamps [12].

Limestone caves are under immense pressure from anthropogenic activities, especially in recent years [9], and are probably one of the centers of biodiversity for certain types of cyanobacteria [2], especially for species from families Hapalosiphonaceae and Symphyonemataceae [43].

photoprotective pigments and bioactive compounds. Rock inhabiting cyanobacteria can be divided into "epilithic," colonizing the substrate surface directly; "hypolithic," living under pebbles and small stones lying on the rock; and "endolithic," living in an upper layer of rock [29]. The cavernicolous cyanobacteria species reported from rock and stone wall surfaces, mostly coccoid and heterocytous types with (often colored) mucilaginous

**Cyanobacteria Origin References** *Hassalia byssoidea* Božana Cave (Serbia) Popović et al. [28] *Leptolyngbya carnea* Gelda Cave (Spain) Martínez & Asencio [21] *Leptolyngbya gracillima* Kastria Cave (Greece) Lamprinou et al. [2] *Leptolyngbya leptotrichiformis* Gelda Cave (Spain) Martínez & Asencio [21] *Lyngbya palikiana* Kastria Cave (Greece) Lamprinou et al. [2]

Czerwik-Marcinkowska et al. [26]

Czerwik-Marcinkowska et al. [26]

Czerwik-Marcinkowska et al. [26]

Czerwik-Marcinkowska et al. [26]

Czerwik-Marcinkowska et al. [26]

Czerwik-Marcinkowska et al. [26]

Martínez & Asencio [21], Czerwik-Marcinkowska et al. [26]

Czerwik-Marcinkowska et al. [26]

Martínez & Asencio [21], Lamprinou et al. [2], Czerwik-Marcinkowska et al. [26]

Lamprinou et al. [2], Popović et al. [28], Czerwik-Marcinkowska et al. [26]

*Nodularia harveyana* Caves from Ojców National Park (Poland)

50 Cyanobacteria

*Nodularia sanguinea* Caves from Ojców National Park (Poland)

*Nostoc commune* Selinits Cave (Greece), Božana Cave

*Nostoc punctiforme* Caves from Ojców National Park (Poland)

*Phormidium breve* Caves from Ojców National Park (Poland)

*Phormidium formosum* Caves from Ojców National Park (Poland)

*Phormidium vulgare* Caves from Ojców National Park (Poland)

*Pseudocapsa dubia* Gelda Cave (Spain), caves from

*Scytonema julianum* Gelda Cave (Spain), Kastria Cave

*Tolypothrix epilithica* Caves from Ojców National Park (Poland)

Park (Poland)

(Serbia), caves from Ojców National

*Pleurocapsa minor* Gelda Cave (Spain) Martínez & Asencio [21]

Ojców National Park (Poland)

(Greece), caves from Ojców National

*Pseudophormidium spelaeoides* Kastria Cave (Greece) Lamprinou et al. [2] *Scytonema drilosiphon* Božana Cave (Serbia) Popović et al. [28]

Park (Poland),

**Table 1.** Cyanobacteria species reported in caves based on literature results since 2010.

*Scytonema mirabile* Božana Cave (Serbia) Popović et al. [28] *Symphyonema cavernicolum* Gelda Cave (Spain) Martínez & Asencio [21] This high diversity may partly be caused by the lack of photosynthetically active radiation which is almost the only stressor in caves, whereas subaerophytic habitats are significantly affected by many stress factors such as excessive irradiance, UV, desiccation, rapid temperature change, and their combinations.

Pentecost and Zhang [48] and Uher and Kovacik [49] noted that type of substratum is an important factor determining the species composition, distribution, and structure of cavernicolous species. They observed that growth of cyanobacteria such as *Anabaena oscillarioides, Gloeocapsa biformis,* and *Nostoc punctiforme* was dependent on the temperature, light, and humidity. These cyanobacteria prefer the humid places during their development, but they also display a considerable resistance to drying as well as to a low air temperature during winter. The adaptation mechanism of cyanobacteria living in a low temperature is not yet precisely known [39], but *Scytonema julianum* is reported as an atmophytic cyanobacterium grown in limestone cave walls in small clusters in shaded vadose settings throughout the

Diversity of Cyanobacteria on Limestone Caves http://dx.doi.org/10.5772/intechopen.79750 53

The cyanobacteria species distribution in relation to cave morphology, lithic substrate, and

An investigation of the diversity and ecology of cyanobacteria in limestone caves has been conducted for many years. Cyanobacteria were the dominant group of phototrophs colonizing cave walls. Chroococcales was the most common cyanobacterial order (with *Gloeocapsa* as the most frequently encountered cyanobacterial genus), followed by Nostocales. The most widespread and abundant species were *Aphanocapsa muscicola, Gloeocapsa biformis,* and *Nostoc commune.* Caves impacted by severe disturbances, including tourism and artificial illumination, were never been completely restored to their former ecological state [47]. Principally, every visitor entering a cave, from the professional speleologist to tourists, has the potential

Department of Botany, Institute of Biology, Jan Kochanowski University, Kielce, Poland

[1] Roldán M, Clavero E, Canals T, Gȯmez-Bolea A, Ariño X, Hernández-Mariné M. Distribution of pototrophic biofilms in cavities (Garraf, Spain). Nova Hedwigia. 2004;**78**:329-351

[2] Lamprinou V, Danielidis DB, Economou-Amilli A, Pantazidou A. Distribution survey of cyanobacteria in three Greek caves of Peloponnese. International Journal of Speleology.

world and is prone to rapid calcification [50].

to exert a negative impact on the cave ecosystem.

Czerwik-Marcinkowska Joanna\* and Massalski Andrzej \*Address all correspondence to: marcinko@kielce.com.pl

**4. Conclusions**

**Author details**

**References**

2012;**41**(2):267-272

microclimatic conditions still remain a challenge for further studies.

It is well known that cyanobacteria are considered the pioneering inhabitants in the caves colonization. Cyanobacteria prevail in the cave entrances compared to the other microalgae [39]; however, they colonize the various parts of the cave entrances, where biodiversity of organisms is the lowest [22]. Among many factors influencing cavernicolous species, water relations in caves are important for cyanobacteria to growth and their colonization [44]. Lamprinou et al. [4] stated that cavernicolous species are dominated by cyanobacteria, which represent the first photosynthetic colonizers on the calcareous surfaces usually thriving both as epiliths and as endoliths. Epilithic communities form extensive dark-green coverings created by *Phormidium breve* as dominant species, or pale blue-green to whitish coverings consisting *Tolypothrix epilithica*. Lamprinou et al. [30] observed predominance of Oscillatoriales group over Chroococcales, in the dim light zone, and also in the entrance, especially in speleothems exposed to light, but their presence is attributed to the chasmoendolithic mode of life.

Round [45] differentiated the distribution of microorganisms depending on the access of either natural or artificial light. Growing of cyanobacteria visible in the form of different color patches on cave walls is undoubtedly connected with the availability of light and specific limestone cave microclimate. This microclimate in caves is influenced by air circulation, hydrological conditions, and isolation of cave from the outside thermal influences [21]. Microscopic observations [21] revealed that cyanobacteria are arranged in particular communities named patinas, which are blue, brown, green, or gray. These communities contain coccoid forms that are frequently accompanied by filamentous forms that are irregularly distributed and do not present stratification. Generally, there are two different areas of the caves. One area is the entrance, where the microclimate is influenced by the light, temperature, and relative humidity fluctuating throughout the year. Patina is greenish-bluish formed by coccoid species only, and there are also grayish patina constituted by coccoid and filamentous species. The second area is the inside with a stable temperature and relative humidity and very low light. The patina found there are greenish-bluish formed by only coccoid species, brownish-gray patina constituted by coccoid forms and filamentous forms, and bluish-grayish patina formed by coccoid forms and filamentous forms. On the other hand, the cave tourism is an important factor causing increase of temperature and environmental changes. Pouličková and Hašler [11] observed the majority caves in Europe are characterized by an average humidity (circ. 70%), and their entrance walls usually are covered by cyanobacteria. The development of cave tourism requires alteration of natural corridors, installation of lighting, pathways, platforms, and associated infrastructure [46]. On the other hand, caves impacted by severe disturbances, including tourism and artificial illumination, have never been completely restored to their former ecological state [47]. Under such conditions, the oligotrophic nature of cave environments is expected to change through organic inputs that alter both the food web and the abundance and distribution of cave organisms [40].

Pentecost and Zhang [48] and Uher and Kovacik [49] noted that type of substratum is an important factor determining the species composition, distribution, and structure of cavernicolous species. They observed that growth of cyanobacteria such as *Anabaena oscillarioides, Gloeocapsa biformis,* and *Nostoc punctiforme* was dependent on the temperature, light, and humidity. These cyanobacteria prefer the humid places during their development, but they also display a considerable resistance to drying as well as to a low air temperature during winter. The adaptation mechanism of cyanobacteria living in a low temperature is not yet precisely known [39], but *Scytonema julianum* is reported as an atmophytic cyanobacterium grown in limestone cave walls in small clusters in shaded vadose settings throughout the world and is prone to rapid calcification [50].

The cyanobacteria species distribution in relation to cave morphology, lithic substrate, and microclimatic conditions still remain a challenge for further studies.
