Exploring microbial diversity in South Korean caves through shotgun sequencing: contrasting dry and wet environments, swabbing versus sediment sampling

1. Introduction

Caves are considered oligotrophic environments due to their limited nutrient and energy sources to support life. Nevertheless bacteria, which constitute the major portion of cave biodiversity, can survive and thrive in this type of environment by modifying their metabolic strategies and microbial communities are also key organisms in the subterranean food web and in decompositions processes (Graening and Brown, 2003). In karstic environments, bacteria are affected by the geology of the surroundings and by the presence of specific geochemical elements and can utilize inorganic compounds for their energetic sustainment (Barton, 2006; De Mandal et al., 2017). Trophic levels and light intensity define the different zonal gradients within a cave and influence the distribution and abundance of the microbiota on the different substrates (Ghosh et al., 2017).

The cave's interior can be categorized into four primary zones based on light penetration: 1) the entrance zone; 2) the twilight zone, characterized by diminishing light until; 3) the transition zone, which remains dark but still experiences variations in temperature and moisture; and 4) the deep zone, where darkness is absolute, humidity is high, and the temperature remains constant (Ghosh et al., 2017).

Since microorganisms need to survive in low nutrient environments, they can use minerals and inorganic matter for catalyzing reactions (Dong et al., 2020) and are also involved in many in-cave mineralization processes. For example, calcifying bacteria promote carbonate precipitation (Cacchio et al., 2003), possibly in the form of speleothems (Cacchio et al., 2004), and/or catalyze the development of crystalline fabrics in stalagmites via metabolic processes or, intrinsically, via water evaporation and CO₂ degassing (Franchi and Frisia, 2020).

On the other hands, heterotrophic microorganisms, including certain bacterial groups and fungi, thrive in caves on the abundant organic matter of substrates like guano, utilizing organic carbon as a source of energy (Dimkić et al., 2021). Since cave substrates are abundant in inorganic molecules such as iron (Parker et al., 2022), manganese (Carmichael et al., 2013), ammonia (Martin-Pozas et al., 2023), methane (Webster et al., 2022) and sulphur (Jones et al., 2016), chemolithoautotrophic organisms are particularly abundant in caves.

To overcome growth limiting factors, microorganisms establish sophisticated, mutualistic networks. This strategy enables a greater number of organisms to survive and grow in the extreme conditions of caves. On cave walls and speleothems, multispecies biofilms consisting of algae/ cyanobacteria, bacteria, or fungi, promoting nutrient circulation enabling diverse organisms to thrive in this harsh environment. Conversely, some bacteria favour competition over cooperation by producing secondary metabolites that suppress the growth of nearby microorganisms, particularly fungi (Roldán et al., 2009; Jaroszewicz et al., 2021; Kosznik-Kwaśnicka et al., 2021).

The main groups described in the cave ecosystem are Actinobacteria, Proteobacteria, Bacteriodetes, Acidobacteria, Firmicutes (Kato et al., 2024). Actinobacteria are able to capture CO₂, which is highly abundant in the cave air and in turn promoting the precipitation of calcium carbonate and the emergence of speleothems (Cuezva et al., 2012).

Proteobacteria, the second largest phyla in caves, thrive in sediments, water, and rocks and can metabolize methane as their sole carbon and energy source (Zhu et al., 2021).

Moreover, heterotrophic bacteria in caves can be pathogenic and may contribute to emerging diseases, such as the Firmicutes phylum that thrive in substrates with high organic content, like close to waste disposal (Tomova et al., 2013) in caves frequently visited by people.

In this study we describe microbial communities from different South Korean caves and explore whether there is a correlation to different environmental conditions. Particularly, we wanted to identify possible differences in microbial composition between dry and wet caves and along the depth-gradient inside the caves. Two types of cave have been chosen: caves not accessible to the public and touristic show-caves, to inspect whether human disturbances impact the composition of bacterial communities.

Different sampling methods can yield varying insights into microbial communities; therefore two types of samples were selected, cave sediments and swabs of surfaces. Swabbing is often used to collect surface-associated microbes, providing information on microbial colonization patterns on cave walls and formations. In contrast, sediment sampling captures microbes residing within the cave substrates.

To have a better insight into the metagenomic reconstructions, shotgun metagenomic DNA sequencing based on two independent methods have been used: PCR-free long-reads sequencing with an Oxford Nanopore Technologies (ONT) MinION sequencer using sediment samples only and short-reads sequencing using an Illumina sequencing platform applied to only swabs. ONT MinION Technology major drawback is the high error rate compared to other sequencing technologies nevertheless, this has been consistently improved over the years with the release of new improved flow cells and more accurate basecalling software (Ciuffreda et al., 2021). At the current time of this work, the read accuracy is at 97%. The Illumina platforms, while performing better in terms of read accuracy and error rate (> 99%), when use short read lengths (150-300 bp) make it difficult to resolve complex genomic regions, such as those containing repetitive elements or large structural variants (Chiang and Dekker, 2020).

South Korean cave systems have not received much international scientific attention. International peer-reviewed studies on South Korean caves have so far focused on the microbial diversity found in moonmilk encrustations from Baeg-nyong Cave (Park et al., 2020), biota found in caves from the Gangwon and Chungcheongbuk provinces (Lee et al., 2020; Chang et al., 2021) and speleothem-based paleoclimate reconstructions by Jo et al. (2010, 2014, 2017). With this paper we aim to add to the knowledge about South Korea as a region of interest for cave systems based environmental reconstructions. Specifically, this work has been intended for paving the way to paleoenvironmental research using stalagmites as a source of ancient DNA (aDNA) and explore their potential as biological paleoarchives. Since the preservation of the aDNA is favored in deposits deeper inside caves (Stahlschmidt et al., 2019), it is crucial to understand the diversity of the local microbial communities and how the in-cave depth gradient plays a role. Some of the caves studied here showed a clearer microbial gradient compared to others but they all seem to agree that relatively drier surfaces are characterized by a preponderance of microbes belonging to the phylum Actinobacteria (also known as Actinomycetes) also confirmed by previous studies (Vardeh et al., 2018; Park et al., 2020).

2. MATERIALS AND METHODS

2.1. Geographical context and description of the caves

The present work examines the microbial communities of five South Korean caves from the central area of the country (Fig. 1) that have been divided into three categories: dry, seasonally wet, and permanently wet.

Fig. 1.

Cave maps with their geographical locations in the Korean peninsula (map from ESRI National Geographic, obtained using QGIS software). Blue outline for Ondal Cave (wet cave), red outline for Baram Cave (dry cave) and black outline for Seodae and Sanjidang Caves (seasonally wet caves). Simbok Cave map is unreported therefore is has been excluded from this figure. Maps have been modified from the originals. Surveyors: Kyoung-nam Jo and Moo-Yeol Lee (Seodae Cave map), Ki-Bok Kim and Sang-Ho Kim (Baram Cave map), Korea Cave Research Institute (Sanjidang and Ondal Cave maps).

2.1.1. Wet cave

Ondal Cave (ON) located in Danyang-gun, Chungcheongbuk-do (37°03.32'N, 128°29.27'E), is a show-cave open all year round to the public excluding during the monsoon season, when it can get flooded (Fig. 1). Like most Korean caves, Ondal does not extend to any considerable depth being developed only for less than 1 km. Air parameters monitoring has been performed in four occasions between November 2020 and April 2022 and showed a constant temperature of ~13℃ in the inner parts of the cave and high CO2 pressure, ranging from 460 ppmv up to 1100 ppmv due to seasonal ventilation processes. The accessible level of the cave corresponds to the phreatic zone with the subterranean river constantly flowing inside the cave and occasionally inundating parts of the cave. The cave is extremely well decorated with a wide range of deposits.

2.1.2. Dry caves

Simbok Cave (SB) (36°47.13'N, 127°57.76'E) is a small cave located in Goesan-gun, Chungcheongbuk-do in central South Korea (Lee et al., 2020). It is formed by horizontal passages accessible to humans in a standing position. During the time of the visiting and the sampling (October 2020), the cave was mostly dry. Hibernating bats were also observed hanging from the ceiling. For this cave no planimetry has been found but its length can be approximated to be nearly 500 meters.

Baram Cave (BA) (37°20.07'N, 128°29.13'E) (“wind cave” in Korean) is in the Gangwon province but further inland compared to Seodae Cave, in the Pyeongchang-gun county (Kim et al., 2018; Chang et al., 2021). The cave is formed by a single long and horizontal duct (~350 m long), which is quite wide and has high ceiling allowing an easy traversing. Towards the end of the cave, red-clay deposits pile up along the edges of the pavement and in the interstices. At the time of the visit, the cave was dry and excluding a few stalactites and broken stalagmites (only a couple of speleothems noticed) the cave is not decorated with any dripstones, and the bare bedrock is exposed. In some part of the cave, it is possible to observe some anticline fold structures and fault planes.

2.1.3. Seasonally wet caves

Seodae Cave (SD) is a cave in Gangwon province located in the southern part of the Seokbyeongsan Formation (37°57'N, 128°94'E) (Choi et al., 2003) (Fig. 1). The access to the cave is technical challenging and requires rope maneuvers. The structure of the system is quite complex with a combination of vertical and horizontal passages that extend for almost 800 meters. At the time of the exploration, the cave was dry, and no speleothem formations have been observed but during the summer monsoon season the drippings restore, suggesting a fast discharge rate of the vadose layer and seasonally modulated environmental conditions.

Sanjidang Cave (SJ) (37°49.04'N, 128°54.23'E) has multi-leveled, narrow and short (~ 520 meters) passages that mostly develop as a deep crack in the bedrock and “crevasses” obstruct the passing along the conduit (Fig. 1). The cave is peculiar because of coralloid deposits that entirely cover the walls in the deep sectors. At the time of the expedition, no air flows have been perceived by the participants, but the nature of coralloid speleothems indicates the occurrence of some sort of air flow or aerosols combined to active dripping (Vanghi et al., 2017), which most likely occurs during the wet season in summer.

2.2. Samples collection and DNA extraction

For each cave, around 20 grams of sediment were collected using a steel spatula decontaminated with bleach between different sampling actions and put into 50 ml polypropylene tubes. Sterile cotton tip swabs were used to sample areas of speleothem surfaces (5 cm²). To maximize the capture of the microbial diversity inside the cave, different speleothem deposits were swabbed, such as stalagmites, stalactites and flowstones. After swabbing the surface, each swab was placed into 2 ml vials filled with Zymo DNA/RNA shield buffer. The wooden sticks of each swab were then broken by hand making sure not to touch the cotton head submerged in the buffer, and the vials were finally closed with a screwtop. Once in the laboratory, the sediment samples were stored at -20℃ while the tubes with the swabs in DNA/RNA shield buffer were stored in the fridge at 5℃.

A total of 40 samples were collected at different depths inside the caves and named with two letters referring to the cave name and a number gradually increasing with increasing depth inside in the cave. For example, ON01 corresponds to a sample that was taken from a location inside Ondal Cave closer to the entrance compared to ON07 that was taken at a greater distance. Total number and labelling of the samples for each cave are indicated in Table 1 and 2.

Table 1.

Information about the samples (tot. 21) that have been sequenced using MinION sequencing technology.

Table 2.

Information about the samples (tot. 19) that have been sequenced using Illumina sequencing technology.

Some samples have biological replicates that have been indicated with a small letter (“a”, “b” or “c”) after the number. In this study, biological replicates refer to samples removed from the same tube of sediment/soil or to a “twin” sterile swab used to swab the same portion of speleothem surface during the cave expeditions but that have been extracted and sequenced in the labs at different moments in time.

DNA, from a total of 21 sediment samples, was extracted in the metagenomics laboratory of Pusan National University (PNU) from 500 mg of sediment using FastDNA SPIN Kit for soil (MP Biomedicals, United States) following the manufacturer’s indication. The DNA has been quantified using a ThermoFisher Qubit-4 fluorometer and the purity defined by ThermoFisher Nanodrop spectrophotometer. 19 swab samples were sent to Zymo Research Corp., Irvine (CA, USA) laboratories and DNA extracted using ZymoBIOMICS-96 MagBead DNA Kit (Zymo Research, Irvine, CA).

3. Results

3.1. MinION samples taxonomic composition

The taxonomical classification led to a mean of 55% of successfully classified reads (Table 1). After filtering for taxa less than 2% to reduce the chances of false positive predictions, the total number of taxa decreased from 37,498 to 15,377. The taxonomy results were then normalized to 100%.

Among the taxa accounting for more than 2% of abundance, the most prominent ones are Proteobacteria and Actinobacteria (also known as Actinomycetes or Actinomycetota) followed by Acidobacteria, Planctomycetes, Chloroflexi, Candidatus Rokubacteria and Verrucomicrobia (Fig. 2). The microbial distributions of each cave are comparable. These communities are predominantly formed by Proteobacteria accounting for 36% and 33% and 30% of the total for Ondal, Sanjidang and Simbok Caves respectively. Actinobacteria presence along Ondal and Sanjidang Caves maintains stable on average at 13% and 9% respectively but in the samples taken near the entrance (ON01) and in deep sectors (ON8, ON11.a and ON11.b) it increases to on average 30% and 18% respectively. Similarly, Simbok Cave hosts relatively more Actinobacteria 21% of the total microbial composition. On a species level this phylum is dominated by a generic species of Actinobacteria and there is not a clear distinction between the three caves, although Ondal wet Cave shows a slight increase of the species Pseudonocardiales with respect to dry and seasonally wet caves (Fig 3a). Acidobacteria are a minor phylum: ranging from 12% in Ondal Cave, 13% in Simbok Cave and 15% in Sanjidang Cave.

Fig. 2.

Bar-plot of the relative abundances of the most common phyla found in the different caves and sequenced using MinION Nanopore technology. The graph below the bar-plot shows the ratio between the major two phyla: Proteobacteria and Actinobacteria. The samples are grouped by type of environment: dry, seasonally wet (“Ss-wet”) and permanently wet (“Wet”) caves and the soil. SB = Simbok Cave, SJ = Sanjidang Cave, ON = Ondal Cave.

Fig. 3.

Compositional barplots normalized to 100% of main Proteobacteria species and Actinobacteria species from inside the caves grouped into dry, seasonally wet (Ss-wet) and wet groups and sequenced with nanopore MinION. Soil samples have been excluded from these plots. The two species of Actinobacteria bacterium, Betaproteobacteria bacterium and Deltaproteobacteria bacterium have been assigned with same colors (pale blue, green and yellow) because they represent generic species of that specific phylum. (Taxa filtering is based on a mean relative abundance greater than 15% and 30% for Actinobacteria species and Proteobacteria species respectively).

Proteobacteria is the dominant phylum found across all samples (Fig. 2). Among this phylum the most dominant species belong to the class of Deltaprotobacteria (also known as Mixococcota) and Betaprotobacteria (Fig. 3b). Betaprotobacteria show an increase in seasonally-wet and wet environments as well as the Gammaproteobacteria belonging to the Chromatiales species. On the contraty, Alphaproteobacteria are more abundant in dry environments compared to seasonally-wet and wet environments. Inside Ondal Cave sediment samples were collected from different locations (Fig. 4). Near the entrance of Ondal Cave, Betaprotobacteria and Alphaproteobacteria species are higher compared to other sites of the cave. Deep sections are mainly composed by the species Chromatiales and species of Deltaproteobacteria whereas species of Betaprotobacteria show a decrease compared to sectors near the entrance.

Fig. 4.

Compositional bar plots normalized to 100% of main Proteobacteria species for the samples from Ondal Cave sequenced with nanopore MinION. Samples are grouped depending on the site inside the cave where they have been collected: entrance, visitors sector and deep sector. The two species of Betaproteobacteria bacterium and Deltaproteobacteria bacterium have been assigned with same colors (green and yellow) because they represent generic species of that specific phylum. (Taxa filtering is based on a mean relative abundance greater than 30%).

3.2. Illumina samples taxonomic composition

The taxonomical classification led to a mean of 55% of successfully classified reads (Table 1). After filtering for taxa less than 2% to reduce the chances of false positive predictions, the total number of taxa decreased from 49,917 to 34,778. The taxonomy results were then normalized to 100%.

The swabs, like the sediment samples sequenced using the Nanopore MinION (section 3.1), mostly show the presence of Proteobacteria and Actinobacteria phyla (Fig. 5) showing an almost equal distribution of these two phyla. In Baram Cave samples, the dominant phylum is Actinobacteria accounting on average for the 65% of the total, whereas Protobacteria only accounts for the 11%. In Ondal Cave, Actinobacteria and Protobacteria abundances are comparable, being on average 35% and 34% of the total respectively. In Seodae and Sanjidang Caves, half of their microbial composition is formed by Proteobacteria, and only 13% on average is formed by Actinobacteria. The third most abundant phylum, Acidobacteria, has a constant abundance of less than 10% among all the swab samples from all the caves.

Fig. 5.

Bar-plot of the relative abundances of the most common phyla found in the different caves and sequenced using with Illumina NovaSeq. The graph below the bar-plot shows the ratio of the major two phyla: Proteobacteria and Actinobacteria, grouped by type of environment: dry, seasonally wet (“Ss-wet”) and permanently wet (“Wet”) caves and the soil. BA = Baram Cave, SD = Seodae Cave, SJ = Sanjidang Cave, ON = Ondal Cave.

A clear distinction in the composition of Proteobacteria and Actinobacteria species is noticeable between dry caves compared to wet and seasonally-wet caves (Fig. 6). Wet and seasonally-wet cave swab colonies mainly host the species Chromatiales (Phylum Gammaproteobacteria), whereas dry cave samples are mostly composed by species of Beta- and Deltaproteobacteria (Fig. 6b).

Fig. 6.

Compositional barplots normalized to 100% of main Proteobacteria species and Actinobacteria species (using cutoff at average abundance of 0.55%) from inside the caves grouped into dry, seasonally wet (Ss-wet) and wet groups and sequenced with Illumina NovaSeq. (Taxa filtering is based on a mean relative abundance greater than 30% for both Actinobacteria species and Proteobacteria species).

Regarding Actinobacteria, all the three categories of caves are different (Fig. 6a). Wet caves mostly host the species Pseudocardiales, for the 75% of the total abundance. Seasonally-wet cave specimen are almost entirely composed by the species Actinomycetia, while the dry caves present a more heterogenous microbial composition formed by the same amount (40%) of Pseudonocardiales and Rubrobacteraceae species and the rest by the species Actinomycetia.

Inside Ondal Cave sediment samples have been collected from different locations (Fig. 7). Near the entrance of the cave and in the sector where visitors are allowed in, the microbial composition is heterogeneous and mainly formed by species of Delta-, Alpha- and Proteobacteria. Deep sections are mainly dominated by the species Chromatiales.

Fig. 7.

Compositional bar plots normalized to 100% of main Proteobacteria species for the samples from Ondal Cave sequenced with Illumina NovaSeq. Samples are grouped depending on the site inside the cave where they have been collected: entrance, visitors sector and deep sector. (Taxa filtering is based on a mean relative abundance greater than 30%).

3.3. Alpha diversity

The Illumina NovaSeq platform generated an average of 27,320,423 raw reads per sample compared to nanopore sequencing that produced an average of 437,510 raw reads per sample (Table 1 and 2). The ONT technology can analyze long fragments of DNA therefore, whereas the DNA sequenced with the NovaSeq had a fixed length of 150 bp, MinION reads have different lengths and their N50 values (weighted median) range from 1991 bp to 6100 bp (Table 1).

The diversity index was calculated on cleaned and filtered data. Considering the different sequencing depths obtained with the two different sequencing technologies, alpha diversity measures are discussed separately.

In general, the communities analyzed using Illumina Novaseq show similar diversity values of the communities analyzed with the MinION (Table 3 and 4). In Ondal Cave, deep-room communities have lower values compared to the entrance. The Shannon values from this study are higher compared to the values from another South Korean cave that ranged between 3 and 4.9 (Park et al., 2020), however they are similar to the values of cave microbiomes described by Ortiz et al. (2013) (USA) and Wiseschart et al. (2019) (Thailand), Addesso et al. (2021) (Italy).

Table 3.

Alpha diversity indexes for group of samples sequenced using MinION.

Table 4.

Alpha diversity indexes for group of samples sequenced using Illumina technology.

4. Discussion

This study aimed at testing whether cave-dwelling microbial communities are sensitive to environmental conditions inside South Korean caves, given their restricted availability of nutrients and the type of substrate. Therefore, since the main purpose of this study is to describe the biodiversity of the samples as a function of their surrounding environment, shotgun sequencing can be more appropriate compared to the 16S sequencing approach.

Shotgun sequencing, in contrast to the 16S ribosomal RNA sequencing method, can classify down to the species level while also aiming to estimate the relative abundances of the taxa within a sample (Tovo et al., 2020). Other advantages of the shotgun method compared to the 16S-amplicons approach include the mitigation of PCR biases, since it does not rely on the amplification of specific DNA target regions and the size of the database (Logares et al., 2014). In addition, the shotgun approach relies on larger and more comprehensive databases compared the amplicon homologue.

The choice of the Kaiju taxonomic classification algorithms has been reasoned on the proteins database used by the program, which are much more conserved with respect to nucleotide sequences (Menzel et al., 2016).

Cave bacterial consortia are influenced by the geology of the area (Zhu et al., 2019) and the geochemical characteristics of the surface but despite being geographically distant, the structure of the microbial communities from all these five South Korean caves is consistent with what is considered the core cave microbiome: Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Chloroflexi, Planctomycetales, Bacteroidetes, Firmicutes, Acidobacteria, Actinobacteria, Nitrospirae, Gemmatimonadetes and Verrucomicrobia (Hershey and Barton, 2018) (Fig. 2 and 5). The source of these organisms is generally attributed to the soil microbiota transported inside the cave by vertical infiltration or surface streams, air currents or other animals (Kováč, 2018). Variations in the composition of microbial communities, both in the outside soil and in the dripping water, are also likely influenced by seasonal environmental parameters. Particularly Rasche et al. (2011) and Yun et al. (2016), showed that with high temperatures the abundance of Alphaproteobacteria, Acidobacteria and Verrucomicrobia decreases, whereas Betaproteobacteria increase. However, once in the cave, the selective pressure of this oligotrophic environment further sorts the bacterial species changing the structure of the original community of microbes (Ortiz et al., 2013; Lavoie et al., 2017) and even filtering contaminant species. The case of Lechuguilla Cave is emblematic as its native bacterial communities remained dominated by the endemic species despite being contaminated by humans’ urine commensal species introduced during some exploration trips (Johnston et al., 2012).

The most abundant bacteria in all the caves studied in this work are Proteobacteria and Actinobacteria, which are also the most common phyla found in many cave environments (Ortiz et al., 2013; Yun et al., 2016; de Mandal et al., 2017; Lavoie et al., 2017; Wiseschart et al., 2019; Zhu et al., 2019). Both phyla are ubiquitous in nature and Proteobacteria also constitute the largest and most diverse group of the Bacteria domain (Kersters et al., 2006).

Among the Proteobacteria phylum, bacteria belonging to the Gammaproteobacteria taxonomical class have been found in high percentages in the swab samples (Fig. 6 and 7). This agrees with Macalady et al. (2006)’s study, who found this type of Proteobacteria particularly dominant in biofilms in an Italian cave as well as with Jurado et al. (2020) work who mostly found Gammaproteobacteria in vermiculations from an Alpine cave. Similarly, in another cave in Slovenia, microbial communities from the walls were mostly dominated by members of Gammaproteobacteria (Pašić et al., 2009).

Proteobacteria have an important role in caves’ nitrogen cycle. Subphyla like Alpha- and Beta- Proteobacteria play critical role in the fixation of nitrogen, where N² is fixated as ammonia (NH₃) so that it can be utilized by other organisms, and they are also involved in the processes of nitrification and denitrification. Other subphyla like Gamma- and Delta- Proteobacteria are instead involved in other biogeochemical cycles, including sulfur and carbon cycles. They contribute to the carbon cycle by breaking down organic matter into simpler molecules that can be utilized by other microorganisms, or they take part in the process of sulphate reduction where sulphur gets recycled making it available for other biological processes, which is critical in anaerobic environment like caves. These processes are important in maintaining the balance of sulphur and nitrogen in cave ecosystems (Holmes et al., 1999; Ojeda et al., 2019; Knief et al., 2003; Macalady et al., 2006, 2008; Zhu et al., 2021, 2022). Some are also crucial in the formation of biofilms on cave surfaces (Engel et al., 2004) and in the processes of calcification of speleothems (Banks et al., 2010).

Actinobacteria have been found in association with pigment-forming communities on the walls of certain caves that are regularly visited by tourists suggesting that this phylum thrives in anthropized environments (Cuezva et al., 2012; Porca et al., 2012; Vautrin et al., 2021). Moreover, some of its species have also presented some pathogenic potential and health concerns for cave visitors (Jurado et al., 2010). Other species instead dominate pristine caves taking advantage of their ability of degrading complex compounds present in the cave, such as in nitrate reduction processes or to fix CO2 (Severino et al., 2019). Cave Actinobacteria, are also considered a potential source of novel bioactive compounds (Axenov-Gibanov et al., 2016; Adam et al., 2018).

The sediment species communities from all the caves in this study, show an increase of Acidobacteria species (Fig. 2) compared to the swabs where their presence is minimal (Fig. 5). This is a very common phylum of bacteria usually present in soils low in nutrients and often observed associated to Proteobacteria (Kielak et al., 2016).

At a species level, the most dominant taxa found on the walls of the caves from this study are Chromatiales (Gammaproteobacteria), Pseudonocardiales and Actinomycetia (Fig. 6 and 7). Similarly, the communities belonging to sediments from remote parts of the caves, showed an increase in Chromatiales Gammaproteobacteria (Fig. 4).

5. Conclusions

The microbial composition identified for the South Korean caves investigated in this study agrees with the core microbiome for karstic environments (Hershey and Barton, 2018). The more common bacteria found in these colonies belong to the phyla Actinobacteria and Proteobacteria. In general, Proteobacteria dominate communities highly exposed to nutrients and animals. Natural sources of organic matter in caves are DOC and colloidal organics from the soil layers above, then transported by subterranean waters, either dripping or floodings. The second source of nutrients comes from plants and animals (Ikner et al., 2007). Proteobacteria that comprise Gamma-, Beta-, Alpha- and Deltaproteobacteria classes, are largely widespread thanks to their capacity of degrading a broad spectrum of organic materials and thus to survive in hostile and nutrients-deprived environments, like caves (Addesso et al., 2021). In either way, Protobacteria not only are associated to high source of organic matter but also to relatively wet and humid environments where nutrients can be easily transported around (Pašić et al., 2009).

In our study we found high abundances of Proteobacteria for all the well decorated caves with active dripping like in the case of Ondal Cave or seasonally wet, during the summer monsoons, caves like Sanjidang and Seodae Caves (Fig. 2 and 5). The only caves with relatively low percentages of Proteobacteria are Baram and Simbok Caves that were dry and dusty during the exploration and lack speleothem deposits (Fig. 2 and 5). The major pattern of Proteobacteria distribution observed in these caves is a relative increase of Gammaproteobacteria in the deep sectors contrary to Alpha- and Beta-Proteobacteria that show a decrease and can be potentially interpret as due to a major nitrogen availability in these parts of the caves, that would need to be confirmed with a metabolic pathways study. A major presence of Actinobacteria relative to Proteobacteria is here interpreted as a proxy of a relatively dry environment, like in the case of Baram (Fig. 5) and Simbok (Fig. 2) Caves. Whether the relative abundance of Rubrobacteraceae and Chromatiales species of bacteria can be regarded as proxy for dry and wet cave conditions respectively, needs to be further explored in the future. In summary, specific microhabitats play a crucial role in controlling the distribution of microbial communities even in the face of an overall resemblance in microbial communities amid geographically distant cave systems.

Data availability statement

The datasets generated and/or analysed in this study are available in the Sequence Read Archive (SRA), hosted by the National Center for Biotechnology Information (NCBI), available with accession number PRJNA946675.

Acknowledgments

This research was supported by the Institute for Basic Science (IBS) IBS-R028-D1. The kaiju taxonomic classification of sequencing reads was partly performed on the IBS/ICCP supercomputer “Aleph,” 1.43 peta flops high-performance Cray XC50-LC Skylake computing system with 18,720 processor cores, 9.59 PB storage, and 43 PB tape archive space. We also acknowledge the support of KREONET. The cave DNA samples were obtained under the license permission number 2023-0023 by the Cultural Heritage Administration of the Republic of Korea. We would like to thank Prof. Jihwan Hwang from the Anti-Microbial Control Laboratory at Pusan National University for the Nanodrop analyses.

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