Biologia 70/8: 9891002, 2015

Section Cellular and Molecular Biology

DOI: 10.1515/biolog-2015-0127

Down into the Earth: microbial diversity of the deepest cave

of the world
Ieva Kieraite-Aleksandrova, Vilius Aleksandrovas & Nomeda Kuisiene*
Department of Microbiology and Biotechnology, Vilnius University, M.K. Chiurlionio 21/27, Vilnius LT-03101, Lithuania;
e-mail: nomeda.kuisiene@gf.vu.lt

Abstract: In our work, microbial diversity of Krubera-Voronja cave was evaluated in the view of the frequency of human
visits in dierent locations as well as the sampling depth. Sampling in this cave was performed at depths of 220 m to 1640 m.
Cultivation-independent method, namely barcoded pyrosequencing of 16S rRNA gene, was used for this analysis. Our results
demonstrated high bacterial diversity at the phylum and genus levels. We have shown that the bacterial diversity at the
phylum level depends on both the sampling depth and the frequency of human visits in Krubera-Voronja cave. Frequently
visited locations were more diverse at the phylum level than the rarely visited branches. The total number of bacterial
genera both per phylum and per sample correlated with the frequency of human visits but not with the sampling depth.
Some genera, found in Krubera-Voronja cave, seem to be absent or very rare in other caves. The present study represents
the rst report on the microbial diversity in Krubera-Voronja cave.
Key words: pyrosequencing; Krubera-Voronja; speleomicrobiology; 16S rRNA gene; deep cave; microbial diversity.

Introduction
Natural underground habitats, represented by caves,
are considered to be nutrient-limited habitats (Jurado
et al. 2010). They are often high in humidity (90
100%), while the low temperature inside remains relatively stable at or near the mean annual surface temperature (Northup & Lavoie 2001). No light penetrates into
caves, but they are rich in extensive areas of mineral
surfaces (Jurado et al. 2010). Therefore, the caves attract considerable attention of microbiologists in terms
of biodiversity and metabolism given the unique environment.
The literature references on microbial communities
in subterranean environments are generally restricted
to the caves found in Spain, Italy, France, Romania, and
the USA (Jurado et al. 2010). Krubera-Voronja cave is
located in Western Caucasus. This cave is considered to
be the deepest known cave its deepest explored point
is 2196 9 m (Sendra & Reboleira 2012). KruberaVoronja cave has been little explored from the biological
point of view. While biospeleological investigations in
Krubera-Voronja cave started a few years ago and gave
the rst knowledge about invertebrate fauna (Sendra &
Reboleira 2012), nothing is known about the microbial
diversity in this deep cave.
Both cultivation-dependent and -independent
methods were previously used for analysis of the microbial diversity in the caves. Fluorescence in situ hy-

bridization, denaturing gradient gel electrophoresis and

cloning of 16S rRNA gene were used for the cultivationindependent studies of microbial diversity in caves
(Bastian et al. 2009; Jones et al. 2014). Metagenomic
approaches as well as whole-genome sequencing were
also applied for analysis of microorganisms from these
unique environments (Gan et al. 2014; Jones et al. 2014;
Kumaresan et al. 2014).
The aim of the present study was to characterize microbial diversity in Krubera-Voronja cave.
We hypothesized that this previously non-investigated
habitat contains unique communities of prokaryotes.
Cultivation-independent method, namely barcoded pyrosequencing of 16S rRNA gene, was used for our analysis.
Material and methods
Site description and sampling
Krubera-Voronja cave is located in the Arabica massif
(43.4184 N 40.3083 E, Western Caucasus). Sampling in this
cave was funded by the National Geographic Society and
was performed from 30th July to 7th August 2012, at depths
of 220 m to 1640 m. The collected sample materials included
soil and clay from cave walls, sediment, speleothems, drinkable water from the underground camps as well as coloured
spots from cave walls. Sampling sites were chosen considering the dierent depth and intensity of human activities. Microbiota from the rarely visited locations was considered to
be the indigenous microbiota of the cave and was compared

* Corresponding author

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Fig. 1. The map of Krubera-Voronja cave showing locations of sampling points (adapted from Ukrainian Speleological Association
2010).

with that from the frequently visited locations possibly containing contaminants associated with human visits. The indigenous microbiota from the dierent depth was supposed
to dier because of the dierent supply of organic carbon
sources. In total, 26 samples were collected from KruberaVoronja cave. Fourteen samples were collected from the frequently visited locations, and 12 samples from rarely visited branches of the cave. Seven samples were from the upper part (220230 m in depth), 2 samples from the middle part (500700 m in depth), and 17 samples from the
lower part (12151640 m in depth) of the cave. All samples were collected using aseptic techniques and placed into
sterile microcentrifuge tubes. The samples were transported
in a cooler and stored at 20 C until DNA extraction. The
sampling sites are shown on the map of Krubera-Voronja
cave (Fig. 1). Characteristics of the sampling sites are listed
in Table 1.
Construction of primers for barcoded pyrosequencing of 16S
rRNA genes
Amplicon fusion primers were constructed for amplication
of bacterial and archaeal 16S rRNA genes. The forward

primer was composed of the 454 Primer A sequence (5-CCA

TCT CAT CCC TGC GTG TCT CCG AC-3), the key sequence (TCAG), a multiplex identier (a barcode) and the
domain-specic forward primer: 27F primer (5-AGA GTT
TGA TCM TGG CTC AG-3) (Studholme et al. 1999) for
Bacteria and S-D-Arch-0519-a-S-15 primer (5-CAG CMG
CCG CGG TAA-3) for Archaea (Klindworth et al. 2013).
The barcodes were selected from the multiplex identier
standard 454 set (Roche). The reverse primer was composed
of the 454 Primer B sequence (5-CCT ATC CCC TGT
GTG CCT TGG CAG TC-3), the key sequence (TCAG)
and the domain-specic reverse primer: 685R primer (5TCT ACG CAT TTC ACC GCT AC-3) for Bacteria and SD-Arch-1048-a-A-17 primer (5-CGR CRG CCA TGY ACC
WC-3) for Archaea (Klindworth et al. 2013). For Bacteria, the expected PCR product size was 678 bp (excluding
454 Primers, the key sequences and the barcodes) and covered V1V4 variable regions (Chakravorty et al. 2007) of
bacterial 16S rRNA gene. For Archaea, the expected PCR
product size was 545 bp (excluding 454 Primers, the key sequences and the barcodes) and covered V4-V6 variable regions (Chakravorty et al. 2007) of archaeal 16S rRNA gene.

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Microbial diversity of Krubera-Voronja cave

991

Table 1. Characteristics of sampling sites.a

Depth (m) T ( C) H (%)

Sample No. Material

R/FVL

220F1
220F2
220F3

Frequently
Frequently
Frequently

220
220
220

3
3
3

69
69
69

Bact
Arch
Bact

Frequently
Rarely

220
230

3
3

69
76

Bact
Bact

Rarely
Rarely

230
230

3
3

76
76

Bact
Arch

500
700
1215
1215
1350
1350
1350
1410
1410
1410
1500
1530
1550
1550
1550
1620
1640
1640
1640

3
4
6
6
6
6
6
8
8
8
6
6
6
6
6
6
6
6
6

>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90

Bact
Bact
Bact
Arch
Arch, Bact
Arch, Bact
Arch, Bact
Bact
Arch, Bact
Arch, Bact
Arch, Bact
Bact
Bact
Bact
Bact
Bact
Arch
Arch, Bact
Bact

220F4
230R1
230R2
230R3
500WF1
700WF1
1215WF1
1215F2
1350R1
1350R2
1350R3
1410WF1
1410F2
1410F3
1500R1
1530R1
1550R1
1550R2
1550R3
1620R1
1640F1
1640F2
1640WF3

Soil (Crimea meander)

Clay from ceiling (Crimea meander)
Deposited carbide from carbide lamps
(Crimea meander)
Green clay (Crimea meander)
White calcite crystals (nonKujbyshevskaja meander)
Soil (non-Kujbyshevskaja meander)
Helictites (non-Kujbyshevskaja
meander)
Water
Water
Water
Soil
Black soil
White clay
Yellow clay
Water
Soil, rust
Black soil
Red spots
White calcite crystals
Irregularly-shaped spots
Soil
Soil
Yellow and red spots
Soil
Soil
Water

Frequently (underground
Frequently (underground
Frequently (underground
Frequently (underground
Rarely visited branch
Rarely visited branch
Rarely visited branch
Frequently (underground
Frequently (underground
Frequently (underground
Rarely
Rarely
Rarely visited ledge
Rarely visited ledge
Rarely visited ledge
Rarely
Frequently (underground
Frequently (underground
Frequently (underground

camp)
camp)
camp)
camp)

camp)
camp)
camp)

camp)
camp)
camp)

DSPA

a R/FVL, rarely/frequently visited location; H, humidity; DSPA, domain-specic primers applied: Arch, Archaea-specic primers;
Bact, Bacteria-specic primers.

Total DNA extraction and amplification of 16S rRNA gene

Total DNA from the samples was extracted using the innuSPEED Soil DNA kit (Analytik Jena AG). For amplication, total DNA from samples 1550R1 and 1550R3 as
well as 1350R1, 1350R2, and 1350R3 (Table 1) was pooled.
Bacterial 16S rRNA genes were amplied in 50 L of reaction mixture containing the DreamTaq Green PCR Master
Mix (2X) (Thermo Fisher Scientic), 0.25 M each primer,
and 10 ng of total DNA. Amplications of bacterial 16S
rRNA gene were conducted under the following conditions:
initial denaturation at 95 C for 2 min followed by 29 cycles each consisting of 95 C for 1 min, 50 C for 2 min and
72 C for 3 min with a nal extension step at 72 C for 7 min
in an Eppendorf thermal cycler. Archaeal 16S rRNA genes
were amplied in 50 L of reaction mixture containing PCR
buer with (NH4 )2 SO4 , 2 mM MgCl2 , 0.2 mM each dNTP,
0.25 mM each primer, 1.25 U recombinant TaqDNA Polymerase (Thermo Fisher Scientic) and 10 ng of total DNA.
Amplications of archaeal 16S rRNA gene were carried out
under the following conditions: initial denaturation at 95 C
for 2 min followed by 29 cycles each consisting of 95 C for
30 s, 50 C for 1 min and 72 C for 1 min with a nal extension step at 72 C for 7 min in an Eppendorf thermal cycler.
Products of amplications were analysed by electrophoresis
through 1% agarose gel. PCR products were puried using
the GeneJET Gel Extraction Kit (Thermo Fisher Scientic).
Sequence analysis
Puried PCR products were subjected for pyrosequencing.
Barcoded pyrosequencing of bacterial 16S rRNA gene was
performed by the LGC Genomics GmbH (Germany) using a GS FLX Titanium platform (Roche). Barcoded pyrosequencing of archaeal 16S rRNA gene was performed

at the University of Cambridge (United Kingdom) using

a GS Junior Plus System (Roche). Detection and removal
of chimeras were performed using de novo algorithm of the
UCHIME program (Edgar et al. 2011). After trimming, the
remaining reads were compared to the Ribosomal Database
Project database via RDP MultiClasier 1.1. at the 80%
condence threshold (Cole et al. 2014). Data were normalized using the total count method (Dillies et al. 2013).
Statistical analysis
Analysis of similarity (ANOSIM) in the statistical package
PRIMER v6 (Clarke 1993) was performed on phylum- and
genus-level pyrosequencing data in order to determine the
dierences in the bacterial communities from the dierent
groups of samples. ANOSIM R values (strength of dissimilarity) of R = 0 indicate a high degree of community similarity, whereas those of R = 1 indicate that the groups
of samples are completely distinct from each other. In all
cases, 999 permutations were used. All statistical tests were
considered signicant at P < 0.05.
BioProject accession number
Pyrosequencing data were deposited in the GenBank Sequence Read Archive (Kodama et al. 2012), BioProject acc.
No. PRJNA269249.

Results
Overview of pyrosequencing results
102114 sequences were obtained from pyrosequencing
of prokaryotic 16S rRNA genes: 96293 sequences using
Bacteria-domain specic primers, and 5821 sequences

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992

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quences could be assigned to the phylum irrespective
of the sampling depth as well as the frequency of human visits. For six samples (230R1, 500WF1, 1350R1R3, 1410F3, 1550R2, and 1640F2) this percentage was
lower: 82.9-96.45% of all the sequences. The sample
220F4 was the outlier: only 50.02% of all the sequences
from this sample were identied as representatives of a
particular phylum.
In total, 24 phyla were identied in the samples
from Krubera-Voronja cave. The dominant phyla were
Proteobacteria and Actinobacteria followed by Firmicutes, Bacteroidetes and Acidobacteria (Fig. 2). The
percentage of other phyla (Armatimonadetes, Chlamydiae, Chlorobi, Chloroexi, Cyanobacteria, Deinococcus-Thermus, Elusimicrobia, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes,
Verrucomicrobia, OD1, OP11, WS3, BRC1, SR1, and
TM7) was below 1%. The unclassied Bacteria constituted 6% of all the sequences. Not a single sequence of
Archaea was detected using Bacteria-domain specic
primers.
For most samples from the rarely visited regions
of the cave 92.6-96.1% of all the sequences could be
assigned to the genus. This percentage was lower for
samples 230R1, 1350R1-R3, and 1620R1 only 85.76,
80.86 and 62.0%, respectively, of the sequences from
these samples could be identied as representatives of a
particular genus. For most samples from the frequently
visited locations of the cave 86.295.0% of all the sequences could be assigned to the genus. The samples
220F4, 500WF1, 700WF1, 1410F3, and 1640F2 were
outliers with 28.4, 52.3, 75.7, 82.7, and 70.4%, respectively, of all the sequences in these samples identied
as the sequences of the particular genera.

Fig. 2. Relative bacterial diversity at the phylum level in KruberaVoronja cave. Combined data from all samples (total) as well as
those from rarely and frequently visited locations of the cave are
shown.

using Archaea-domain specic primers. After trimming

(removal of the barcodes as well as low-quality and
short sequence reads), 68967 high-quality sequences
were obtained for 26 samples of Krubera-Voronja cave.
General information about bacterial diversity
in Krubera-Voronja cave
In the majority of samples, 97.3699.54% of all the se-

Bacterial diversity at the rarely visited locations of

Krubera-Voronja cave
In total, 16S rRNA gene sequences of 20 phyla were
identied in the samples from the rarely visited locations. Combined data from all the samples from the
rarely visited locations of the cave showed the dominant position of Proteobacteria followed by Actinobacteria, Firmicutes, unclassied Bacteria, and Acidobacteria (Fig. 2). Bacteroidetes constituted 2% of all the
sequences similar as did the sequences from the remaining phyla. The sequences of Proteobacteria, Actinobacteria, Firmicutes, Acidobacteria and Cyanobacteria as
well as those of unclassied Bacteria were detected
in all samples. Bacteroidetes were also ubiquitous, except for samples 1530R1 and 1550R1+R3. Other phyla
(Armatimonadetes, Chlorobi, Chloroexi, DeinococcusThermus, Elusimicrobia, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes, Verrucomicrobia, as
well as candidate phyla WS3, OP11 and OD1) were
identied in 15 samples from the rarely visited locations of the cave, from 0.02% to 3.14% of all the sequences per phylum in dierent samples.
Because of the dierences in temperature, humidity as well as the supply of organic carbon in the upper
and lower parts of the cave, we chose to compare py-

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Fig. 3. Rarely visited locations of Krubera-Voronja cave: comparison of bacterial diversity at the phylum level in the upper (230 m
in depth) and lower (13501620 m in depth) parts of the cave. Only those phyla that constituted 1% of all the sequences in the
combined data sets are shown.
Table 2. The number of genera from dierent phyla in the samples of Krubera-Voronja cave.a
Sample

Aci Act Arm Bac Chl Chr Chf Cya D-T Elu Fir Fus Gem Nit Pla Pro Spi Ver BRC1 OD1 OP11 SR1 TM7 WS3

RVL, UPC, S
230R1
10
230R2
3
LPC, S
1350R1-R3 15
1500R1
7
1530R1
2
1550R1+R3
1
1550R2
1
1620R1
13
FVL, UPC, S
220F1
11
220F3
1
220F4
7
MPC, W
500WF1
13
700WF1
2
LPC, S
1410F2
9
1410F3
8
1640F2
5
LPC, W
1215WF1
3
1410WF1
9
1640WF3
14

31
29

5
8

31
17
11
18
19
27

2
9

1
12

20
26
13

22
21

13
8

59
21
15

4
2
1

27
10
9

13
17
38

3
2

6
11
25

1
1

1
1

21
28

56
47

1
1
1
2
1
1

1
1

1 19
17
11
16
13
18

1
1

4
1

2
2

26
48
14
10
14
74

35
28
17

16
3
6

2
1
1

1
1

1
1

1
1

23
21

7
4

1
1

36
8
10

3
1

1
1
1
5

1
1
1

1
2
1

20
25
34

1
2

56
33
31

1
1

70
57

1
1
1

1
1

8
7

94
44
41

7
5

1 48
3 45
3 101

1
1
5

1
1
1

1
1

1
1

1
1

1
1

1
1
1

1
1

RVL, rarely visited locations; FVL, frequently visited locations; UPC, upper part of the cave; LPC, lower part of the cave; MPC,
middle part of the cave; S, sediment; W, water. Aci, Acidobacteria; Act, Actinobacteria; Arm, Armatimonadetes; Bac, Bacteroidetes;
Chl, Chlamydiae; Chr, Chlorobi; Chf, Chloroexi; Cya, Cyanobacteria; D-T, Deinococcus-Thermus; Elu, Elusimicrobia; Fir, Firmicutes;
Fus, Fusobacteria; Gem, Gemmatimonadetes; Nit, Nitrospirae; Pla, Planctomycetes; Pro, Proteobacteria; Spi, Spirochaetes; Ver,
Verrucomicrobia.

rosequencing results for the upper part samples (230R1R2) and those for the lower part (1350R1-R3, 1500R1,
1530R1, 1550R1-R3, and 1620R1) (Fig. 3). Statistical
analysis showed that the bacterial community in the
upper part of the cave signicantly diered from that
in the lower part (ANOSIM R = 0.665; P < 0.05).
Our analysis showed that Actinobacteria (46%) were
prevalent in the upper part, followed by Proteobacteria,
Firmicutes, unclassied Bacteria, Acidobacteria, Bac-

teroidetes, and Cyanobacteria. Other phyla constituted

only 0.2% of all the sequences in the upper part and are
not shown in Figure 3. In the lower part of the cave,
Proteobacteria sequences (64%) were the most abundant. The percentage of the sequences of Actinobacteria
and Firmicutes was smaller, and that of Bacteroidetes
was higher in the lower part of the cave than in the upper one. In contrast with the upper part, Chloroexi,
Nitrospirae and Planctomycetes each constituted 1%

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Table 3. The most abundant bacterial genera in the samples from the rarely visited locations of Krubera-Voronja cave.a
Samples

TNG Actinobacteria

Proteobacteria

Firmicutes

Other

Gp3 (3.62; Acidobacteria)

UPC, S
230R1

135 Microbacterium (42.64),

Micrococcus (2.69),
Corynebacterium (1.64),
Propionibacterium (1.26)

Pseudomonas (7.42), Escherichia (2.74), Acinetobacter (1.98)

Staphylococcus (3.94),
Planomicrobium (1.83),
Geobacillus (1.71), Streptococcus (1.21)

230R2

117 Propionibacterium
(21.44), Micrococcus
(3.91), Nesterenkonia
(3.35), Corynebacterium
(2.80)

Halomonas (14.40),
Herbaspirillum (11.42),
Escherichia (8.81),
Acinetobacter (2.31)

Aeribacillus (7.0), Staphylococcus (2.75), Caldalkalibacillus (2.02), Lactobacillus (2.02), Bacillus

(1.11)

1350R1-R3

114 Arthrobacter (3.32),

Nesterenkonia (1.96),
Propionibacterium (1.42)

Halomonas (4.15),
Psychrobacter (2.85),
Herbaspirillum (1.90),
Escherichia (1.54),
Albidiferax (1.36)

Aeribacillus (3.32), Bacil- Gp6 (4.27; Acidobactelus (2.31), Caldalkalibacil- ria), Nitrospira (2.90; Nilus (1.42)
trospirae), WS3 generaincertae sedis (1.96;
WS3), Gp4 (1.78; Acidobacteria), Gp16 (1.66;
Acidobacteria), Gp17
(1.42; Acidobacteria),
Gemmatimonas (1.30;
Gemmatimonadetes)

1500R1

105

Pseudomonas (65.80),
Acinetobacter (6.70),
Perlucidibaca (1.91)

Staphylococcus (2.31),
Aerococcus (1.77)

1530R1

40

Nesterenkonia (13.65),
Propionibacterium (4.80),
Arthrobacter (2.95),
Corynebacterium (1.48),
Pseudonocardia (1.11),
Rothia (1.11)

Herbaspirillum (17.71),
Halomonas (13.65),
Acinetobacter (9.23),
Pseudomonas (1.48),
Psychrobacter (1.11)

Caldalkalibacillus (8.12),
Aeribacillus (4.06),
Staphylococcus (1.48)

1550R1+R3

47

Propionibacterium
(10.72), Arthrobacter
(8.48), Micrococcus
(7.98), Corynebacterium
(3.99), Brachybacterium
(1.75), Nesterenkonia
(1.20)

Psychrobacter (20.45),
Halomonas (4.99),
Herbaspirillum (3.24),
Acinetobacter (1.99),
Enhydrobacter (1.50)

Lysinibacillus (2.99),
Solibacillus (2.24), Paenibacillus (2.0), Bacillus
(1.75), Staphylococcus
(1.50), Planococcaceaeincertae sedis (1.25)

1550R2

49

Propionibacterium
(18.39), Micrococcus
(10.32), Corynebacterium (6.45), Nesterenkonia (4.84), Arthrobacter
(3.23), Brachybacterium
(2.58)

Halomonas (6.77),
Herbaspirillum (3.22),
Psychrobacter (1.61),
Acinetobacter (1.29)

Aeribacillus (6.13),
Geobacillus (3.55), Bacillus (2.90), Caldalkalibacillus (1.94), Lysinibacillus (1.94), Lactobacillus
(1.61), Staphylococcus
(1.29)

1620R1

165 Arthrobacter (3.71),

Pseudonocardia (1.14)

LPC, S

Pseudomonas (32.18),
Escherichia (11.20), Albidiferax (3.55), Acinetobacter (2.76), Hydrogenophaga (2.21), Nitrosospira (1.18), Pseudoxanthomonas (1.18),
Massilia (1.14)

Flavobacterium (3.33;
Bacteroidetes)

Flavobacterium
(2.13; Bacteroidetes),
Ohtaekwangia (1.42;
Bacteroidetes)

a TNG, total number of genera; UPC, upper part of the cave; LPC, lower part of the cave; S, sediment. Numbers in parentheses
indicate the percentage of the sequences of the particular genus. Only genera that comprise 1% of all the sequences per sample are
shown.

of all the sequences, and the remaining phyla comprised further 2% in the lower part of the cave. Phyla

Chlorobi, Elusimicrobia, Spirochaetes, and candidate

phylum OD1 were not detected in the upper part of the

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Microbial diversity of Krubera-Voronja cave

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Fig. 4. Frequently visited locations of Krubera-Voronja cave: comparison of bacterial diversity at the phylum level in the upper (220
m in depth), middle (500700 m in depth) and lower (12151640 m in depth) parts of the cave. Only those phyla that constituted
1% of all the sequences in the combined data sets are shown.

cave, but were identied in the samples from the lower

part.
The number of genera per phylum ranged from 1
to 74 in dierent samples from the rarely visited locations (Table 2). The highest number of genera was identied for Proteobacteria (samples 230R1-R2, 1500R1,
and 1620R1) and Actinobacteria (samples 1350R1-R3
and 1550R1-R3). The total number of genera varied
from 40 to 165 genera per sample (Table 3), and this
number did not depend on the sampling depth. The
most numerous genera, i.e. the genera that comprised
1% of all the sequences per sample mainly belonged
to Actinobacteria, Proteobacteria and Firmicutes (Table 3). The genera of Bacteroidetes were also identied in samples 1500R1 and 1620R1, and those of Acidobacteria were found in samples 230R1 and 1350R1R3. Representatives of the genera of Nitrospirae, WS3

and Gemmatimonadetes were also found in the sample

1350R1-R3.
Bacterial diversity at the frequently visited locations of
Krubera-Voronja cave
Sequences of 23 bacterial phyla were obtained from 11
samples collected at the frequently visited (including
the underground camps) locations of Krubera-Voronja
cave. Combined data from all the samples from the frequently visited locations of the cave showed the dominant position of Proteobacteria (42%) (Fig. 2). The
16S rRNA genes of Actinobacteria, Firmicutes, Proteobacteria, Acidobacteria and Bacteroidetes as well as
those of unclassied Bacteria were detected in all samples. Chloroexi and Cyanobacteria were also ubiquitous, except for sample 220F3 (without Chloroexi) and
sample 1640F2 (without Cyanobacteria). The minor

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Fig. 5. Comparison of bacterial diversity at the phylum level in the water and sediments from the frequently visited locations of
Krubera-Voronja cave. Only those phyla that constituted 1% of all the sequences in the combined data sets are shown.

phyla, such as Armatimonadetes, Chlorobi, Chlamydiae, Deinococcus-Thermus, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes,
Verrucomicrobia, as well as candidate phyla WS3,
OP11, OD1, BRC1, SR1 and TM7 were identied in
1-9 samples from the frequently visited locations of
Krubera-Voronja cave. They constituted from 0.01% to
1.44% of all the sequences per phylum in the dierent
samples.
ANOSIM showed that the bacterial communities
from the upper (220 m), middle (500700 m) and lower
(12151640 m) parts of the cave were signicantly different (Ruppermiddle = 0.332, Rupperlower = 0.291,
Rmiddlelower = 0.316; P < 0.05). The phylum Proteobacteria was the dominant phylum (3344% of all
the sequences) in the frequently visited places irrespective of the sampling depth (Fig. 4). While Actinobacteria followed Proteobacteria in the samples from both
the upper and the lower parts of the cave, phylum Firmicutes was the second numerous taxon in the middle
part of the cave. On the other hand, the percentage of
Firmicutes sequences was similar in both the upper and
the middle parts of the cave, and this was in contrast
with the small percentage of this phylum in the lower
part (Fig. 4). Unclassied Bacteria comprised 1114%
in the upper and middle parts, while they constituted
only 3% in the lower part of the cave. The percentage
of the sequences of Chloroexi and WS3 overpassed 1%
threshold only in the samples collected in the middle
part of the cave (Fig. 4). The phylum level diversity
was the smallest in the upper part Chlamydiae, Fusobacteria, and Spirochaetes were not found in this part
of the cave, while were identied in both the middle and
lower parts. In addition, Chlorobi and SR1 as well as
BRC1 and TM7 were not detected in the upper part

of the cave, while they were identied in the middle

and lower parts, respectively. The interesting trend was
observed for Bacteroidetes the percentage of their sequences increased from the upper to the lower part of
the cave from 2% at a depth of 220 m to 9% at a depth
of 12151640 m (Fig. 4).
As the samples of both water and sediments were
collected in the frequently visited places, the analysis water vs. sediments was carried out. Our analysis showed that these two bacterial communities were
signicantly dierent (ANOSIM R = 0.536; P < 0.05).
The main dierence between both types of the samples
was determined to be the percentage of the sequences of
Actinobacteria and Proteobacteria (Fig. 5). The dominant position of Actinobacteria in sediments and not
in water could have been expected these bacteria
are considered to be terrestrial bacteria. Our analysis
showed that the percentage of Bacteroidetes sequences
did not depend on the type of the sample and was equal
in both water and sediments to be 7%. Some of Bacteroidetes, namely Bacteroidales, are known to be excellent indicators of faecal pollution (McLellan & Eren
2014). Our analysis of the samples from the frequently
visited locations (including the underground camps)
revealed that Bacteroidales were present as only 13
sequences per sample if any. Flavobacteriales was the
prevalent order of Bacteroidetes (data not shown), and
Flavobacterium sequences were among the most numerous genera in the frequently visited regions of the cave
(Table 4).
The number of genera per phylum was from 1 to
101 in the dierent samples from the frequently visited locations (Table 2). The highest number of genera
was identied for Proteobacteria in all samples. The
total number of genera varied from 82 to 259 genera

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Table 4. The most abundant bacterial genera in the samples from the frequently visited locations of Krubera-Voronja cave.a
Samples

TNG Actinobacteria

Proteobacteria

Firmicutes

Other

Gp3 (4.88; Acidobacteria),

Sediminibacterium (1.83;
Bacteroidetes)

UPC, S
220F1

156

Microbacterium (23.58),
Propionibacterium (2.55),
Corynebacterium (2.03),
Micrococcus (1.80)

Massilia (6.23), Pseudomonas (4.85),

Halomonas (4.28),
Stenotrophomonas (3.18),
Escherichia (3.03), Acinetobacter (2.65), Sphingomonas (1.08)

Streptococcus (3.50),
Aeribacillus (2.60),
Staphylococcus (2.35),
Geobacillus (1.25)

220F3

93

Propionibacterium (8.92),
Nesterenkonia (5.74),
Corynebacterium (3.28),
Micrococcus (3.14),
Arthrobacter (1.59)

Halomonas (20.66),
Herbaspirillum (11.38),
Escherichia (2.73), Acinetobacter (1.59), Psychrobacter (1.18)

Aeribacillus (11.52), Caldalkalibacillus (4.23),

Bacillus (2.05), Staphylococcus (1.96), Geobacillus (1.37), Oceanobacillus (1.23), Lactobacillus
(1.18)

220F4

82

Propionibacterium (1.43),
Corynebacterium (1.10),
Micrococcus (1.0)

Pseudomonas (8.74),
Acinetobacter (1.76),
Halomonas (1.43)

Planomicrobium (1.72),
Exiguobacterium (1.05)

Pseudomonas (17.18), Albidiferax (2.66), Paracoccus (1.26)

Planomicrobium (13.59),
Exiguobacterium (7.04),
Staphylococcus (2.39)

MPC, W
500WF1

174

Gp6 (2.93; Acidobacteria), WS3 generaincertae sedis (1.29; WS3),

Flavobacterium (4.70;
Bacteroidetes)

700WF1

113

Propionibacterium (2.45),
Corynebacterium (1.71),
Micrococcus (1.43)

Escherichia (34.26), Pseu- Staphylococcus (2.45),

domonas (6.27), AcineStreptococcus (1.14)
tobacter (4.50), Massilia
(3.25), Citrobacter (3.14),
Alkanindiges (1.08)

1410F2

259

Arthrobacter (30.88), Humicoccus (2.01), Salinibacterium (1.87), Propionibacterium (1.62),

Cryobacterium (1.0)

Albidiferax (3.75), Pseudoxanthomonas (2.41),

Herbaspirillum (1.88),
Polaromonas (1.64),
Halomonas (1.61), Psychrobacter (1.36), Acinetobacter (1.28), Pseudomonas (1.15)

Trichococcus (4.76)

Flavobacterium (3.41;
Bacteroidetes), Gp4 (2.69;
Acidobacteria)

1410F3

114

Ilumatobacter (2.77)

Pseudomonas (8.20),
Haliea (6.31), Rhizobacter
(2.37), Halomonas (1.13)

Pasteuria (1.57)

Ferruginibacter (18.05;
Bacteroidetes), Terrimonas (3.05; Bacteroidetes), Gp6 (4.26;
Acidobacteria), Haloferula
(1.93; Verrucomicrobia),
Gemmata (1.45; Planctomycetes), Gp4 (1.29;
Acidobacteria)

1640F2

85

Ilumatobacter (4.64)

Acinetobacter (11.59),
Aquicella (7.32), Pseudomonas (3.48), Nitrosococcus (2.32), Enhydrobacter (1.45),
Escherichia (1.30),
Halomonas (1.23), Pseudoxanthomonas (1.16),
Massilia (1.16), Legionella
(1.09)

LPC, S

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OP11 genera-incertae
sedis (1.23; OP11)

998

I. Kieraite-Aleksandrova et al.

Table 4. (continued)
Samples

TNG Actinobacteria

Proteobacteria

Firmicutes

Other

W
1215WF1

98

Pseudomonas (59.08), Escherichia (16.81), Acinetobacter (2.72), Citrobacter

(1.4)

1410WF1

122

Propionibacterium (8.49),
Micrococcus (3.70),
Corynebacterium (2.86),
Nesterenkonia (1.01)

Acinetobacter (13.20),
Halomonas (5.80), Pseudomonas (5.63), Escherichia (3.28), Massilia (2.69), Rheinheimera
(2.10), Stenotrophomonas
(2.10), Cellvibrio (1.26),
Enhydrobacter (1.10)

1640WF3

235

Propionibacterium (6.68),
Arthrobacter (6.17)

Albidiferax (4.86),
Acinetobacter (3.40),
Dechloromonas (2.80),
Alkanindiges (2.0), Rugamonas (1.32), Polaromonas (1.28), Massilia
(1.24), Halomonas (1.08)

Flavobacterium (1.18;
Bacteroidetes)

Aeribacillus (2.52), Streptococcus (1.60), Staphylococcus (1.60)

Chryseobacterium
(6.73; Bacteroidetes),
Wautersiella (2.35;
Bacteroidetes),
Flavobacterium (1.68;
Bacteroidetes)

Flavobacterium (10.18;
Bacteroidetes)

a TNG, total number of genera; UPC, upper part of the cave; MPC, middle part of the cave; LPC, lower part of the cave; S, sediment;
W, water. Numbers in parentheses indicate the percentage of the sequences of the particular genus. Only genera that comprise 1%
of all the sequences per sample are shown.

per sample, and this number did not depend on the

sampling depth (Table 4). Similarly to the results for
the rarely visited locations, Actinobacteria, Proteobacteria and Firmicutes comprised the majority of the
genera with 1% of all the sequences per sample in
the frequently visited branches (Table 4). The genera
of Bacteroidetes were also abundant the sequences
of the genera from this phylum (1% of all the sequences per sample) were identied in all the samples
except for 220F3-F4, 700WF1, and 1640F2. The genera of Acidobacteria, Verrucomicrobia, Planctomycetes,
OP11, and WS3 were also identied in some samples
(Table 4). The abundance of some genera depending
on the sampling depth is noteworthy. For example, Microbacterium was one of the most abundant genera only
in the upper part of the cave (sample 220F1; Table 4),
and not only in the frequently visited locations (sample
230R1; Table 3).
Bacterial diversity in the dierent types of samples
The inuence of the nature of the sample (soil
vs. speleothems vs. coloured spots from the cave
walls) on the bacterial diversity was also evaluated
(Fig. 6). The sampling depth as well as the frequency of human visits were disregarded in this analysis. ANOSIM showed that the bacterial communities
from these three types of the samples diered significantly (Rsoilspeleothems = 0.485, Rsoilspots = 0.622,
Rspeleothemsspots = 0.591; P < 0.05). Acidobacteria,
Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria as well as unclassied Bacteria were detected in
all three types of samples. Planctomycetes and Chloroexi constituted 1% of all 16S rRNA gene sequences

in soil samples but not in speleothems and coloured

spots.
The percentage of Actinobacteria, Firmicutes, and
Proteobacteria was the main dierence between soil
samples, speleothems and samples from coloured spots.
While phylum Actinobacteria was the most numerous
(54% of all sequences) in speleothems, it constituted
only 7% in the samples from coloured spots. In contrast, phylum Proteobacteria was the absolute leader
in the latter samples 79% of all sequences. The percentage of Firmicutes was similar in soil samples and
speleothems (11 and 13%, respectively), but this phylum was among the less numerous (4%) in the samples
from the spots.
Archaeal diversity in Krubera-Voronja cave
Total DNA from 11 samples was used as a template
in PCR with the Archaea-domain-specic primers (Table 1). Only a single archaeal 16S rRNA gene sequence
was obtained from the three samples: 16S rRNA gene
sequence of Creanarchaeota from samples 1500R1 and
230R3, and that of unclassied Archaea from sample
1640F1. The larger set of archaeal sequences (24 sequences) was obtained only from samples 1350R1-R3.
Out of these 24 sequences, 15 sequences were found to
belong to unclassied Archaea, 7 sequences to Crenarchaeota, and 2 sequences to Euryarchaeota. All crenarchaeal 16S rRNA gene sequences were identied as unclassied Thermoprotei. Euryarchaeal 16S rRNA gene
sequences were identied as unclassied Euryarchaeota.
At the initial step of our work we used Archaeadomain-specic primer pair S-D-Arch-0519-a-S-15/SDArch-1041-a-A-18 recommended by Klindworth et al.

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Microbial diversity of Krubera-Voronja cave

999
D-Arch-0519-a-S-15/S-D-Arch-1048-a-A-17 primer pair
for the further work. PCR product of the correct
size was obtained in this case. But pyrosequencing of
this PCR product showed that it was highly unspecic. As mentioned above, the coverage of archaeal
16S rDNA sequences was extremely low. In contrast,
the coverage of bacterial gene sequences was very
high approximately 97% of all sequences. The sequences of phyla Actinobacteria, Firmicutes (Bacilli,
Clostridia and Erysipelotrichia), Proteobacteria (and -Proteobacteria) and Tenericutes constituted the
majority of bacterial sequences, but 16S rRNA genes of
Acidobacteria, Armatimonadetes, Bacteroidetes, Chloroexi, Cyanobacteria, Planctomycetes, Spirochaetes,
Synergistetes, Thermotogae and Verrucomicrobia were
also detected. Such baing results impelled us to
verify the S-D-Arch-0519-a-S-15/S-D-Arch-1048-a-A17 primer pair using Archaea-rich community from the
anaerobic digester. In total, 6615 sequences were obtained, but methanogenic Euryarchaeota constituted
only 14.36%. Other sequences belonged to those of
Firmicutes (68.86%), unclassied Bacteria (15.93%),
Tenericutes (0.47%), and Actinobacteria (0.21%); a few
sequences were also obtained for Bacteroidetes, Proteobacteria, Spirochaetes, Synergistetes and Thermotogae.
Discussion

Fig. 6. Comparison of bacterial diversity at the phylum level in

the samples of the dierent nature. Only those phyla that constituted 1% of all the sequences in the combined data sets are
shown.

(2013) as the most suitable primer pair for sequencing technologies like Roches 454. Unfortunately, we
could not obtain PCR product of the correct size
using this primer pair. Therefore, we chose the S-

Bacterial diversity of the deepest cave of the world

Based on the published studies reporting 16S rRNA
gene sequences from cultures or clone libraries, roughly
half of the recognized bacterial phyla have been identied in caves (Engel 2010). Not all of these phyla
were detected in the particular cave, i.e. dierent numbers of bacterial phyla were detected in dierent caves.
The number of the detected phyla ranged from 6
(Schabereiter-Gurtner et al. 2002) to 13 (Northup et
al. 2011). In the samples from Krubera-Voronja cave,
24 bacterial phyla were found. To our knowledge, such
high phylum level diversity has never been established
in the caves. On the other hand, no unique phylum was
found in the samples from Krubera-Voronja cave, all
24 phyla have been previously detected in other caves
(Engel 2010). The core set of the phyla, i.e. the phyla
found in all samples from Krubera-Voronja cave, was
composed of Acidobacteria, Actinobacteria, Firmicutes,
and Proteobacteria. These phyla were also the most numerous in all samples. Bacteroidetes were also among
the most abundant phyla albeit they were detected not
in all samples.
A general trend in caves is that Proteobacteria
form the largest group of bacteria (Bastian et al. 2009),
and our study of the whole microbiome of KruberaVoronja cave also matched this tendency. Our analysis
of the combined data also showed that the percentage of
the four prevalent phyla was highly similar independent
of the frequency of human visits. It should be noted that
ANOSIM analysis of the combined data sets revealed
that the bacterial community of the rarely visited loca-

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1000

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tions, i.e. the indigenous microbiota of the cave, signicantly diered from that of the frequently visited locations (R = 0.315; P < 0.05). For the fth phylum, Bacteroidetes, and for the minor phyla Chloroexi, Planctomycetes, and Verrucomicrobia, the other trend was
observed the larger percentage of these phyla was established in the frequently visited locations of the cave.
Namely, these locations were also more diverse at the
phylum level than the rarely visited branches. The differences at the phylum level diversity between these
two types of the samples were associated with the presence/absence of the minor phyla Chlamydiae, Elusimicrobia, Fusobacteria, BRC1, SR1, and TM7.
As only microbiomes of a quite shallow caves were
studied untill now, it was impossible to investigate the
dierences in the bacterial diversity depending on the
sampling depth. But the depth of Krubera-Voronja cave
allowed us to do this research. Our results denitely
showed that bacterial diversity at the phylum level depended on the sampling depth. The change of the rst
prevalent phylum in the samples from the rarely visited
locations was the most surprising. While phylum Proteobacteria was the most abundant in the lower parts
of the cave, Actinobacteria became the rst dominant
in the upper ones. Although this is in contrast with the
phylum level results for other caves (Porter et al. 2009;
Kumaresan et al. 2014), it can be explained by the differences in the organic carbon supply in the upper and
lower parts of the cave: Actinobacteria are typical heterotrophs and, consequently, they thrive in the organic
carbon-rich upper part. This tendency was supposed to
be disturbed by the frequent human visits in the upper
part of the cave, and Proteobacteria became the rst
prevalent phylum in the frequently visited locations in
this part of the cave. The percentage of Firmicutes, that
are usually also heterotrophic bacteria, decreased with
a depth and it was the smallest in the lower part of the
cave, while the percentage of Bacteroidetes, in contrast,
was the smallest in the upper part and increased with
a sampling depth irrespective of the frequency of the
visits. Our results also clearly showed that the phylum
level diversity is the smallest in the upper part of the
cave irrespective of the frequency of human visits.
Analysis of the samples of dierent nature showed
that Proteobacteria was the most numerous phylum in
the samples from the coloured (red and yellow) spots.
This phylum also dominated in the samples from yellow
and grey (Portillo et al. 2008) as well as white (Portillo
et al. 2009) colonisations collected from the walls of Altamira cave. In contrast, Cuezva et al. (2012) reported
that the grey spots collected from the walls and ceiling of the same Altamira cave were composed of Actinobacteria and calcium carbonate crystals. Percentage
of Firmicutes in the samples collected from the coloured
spots in Krubera-Voronja cave was low, and these results were in accordance with those obtained for yellow
and grey colonisations from Altamira cave (Portillo et
al. 2008).
Phylum Actinobacteria was the most numerous in
the samples from Krubera-Voronja caves speleothems.

It should be noted that phyla Acidobacteria, Actinobacteria, and Proteobacteria were revealed to dominate
in bacterial communities in the dierent samples from
speleothems of Kartchner Caverns (Ortiz et al. 2013).
In accordance with the phylum level diversity, the
most numerous genera belonged mainly to Actinobacteria, Proteobacteria and Firmicutes, irrespective of the
frequency of human visits. The total number of the genera both per phylum and per sample correlated with
the frequency of human visits it was higher in the frequently visited locations than in the rarely visited ones.
Our ndings that the frequent visits resulted in higher
diversity at both phylum and genus level in KruberaVoronja cave are in contrast with the data reported
previously, when it was shown that the microbial diversity in caves generally decreases with the human impact
increase (Bastian et al. 2009). On the other hand, it was
hypothesized by Chan & Chong (2014) that the unusual
diversity and abundance of unclassied bacteria in the
coastal waters is associated namely with anthropogenic
activities.
In our work the total number of genera per sample
did not depend on the sampling depth. But the abundance of some genera to a certain degree depended on it.
For example, although the genus Microbacterium was
identied in all samples except for 1530R1 (data not
shown), it was the most abundant only in two samples
from the upper part of the cave. Although identied
in the samples from the dierent sampling depth (data
not shown), Enhydrobacter, Ilumatobacter, and Pseudoxanthomonas were numerous only in the lower part
of the cave albeit not in all the samples.
Some actinobacterial genera (Nocardia, Mycobacterium, Gordonia, Rhodococcus, and Streptomyces)
were reported to be widely spread in the caves (Jurado et al. 2010), but these genera were not among
the most numerous in Krubera-Voronja cave. Instead of
them, actinobacterial genera Arthrobacter, Corynebacterium, Micrococcus, Nesterenkonia, and Propionibacterium were ubiquitous in all samples as well as
among the most numerous genera in the majority
of the samples. These genera as well as Brachybacterium, Microbacterium, Pseudonocardia, and Rothia
were previously also detected in other caves (Groth
et al. 2001; Portillo et al. 2009; Grin et al. 2014).
Out of Firmicutes, only Staphylococcus was found in
all the samples. This genus as well as Aerococcus,
Bacillus, Geobacillus, Paenibacillus, and Streptococcus were also detected in other caves (Groth et al.
2001; Engel 2010; Grin et al. 2014). Other genera
of Gram-positive bacteria (Cryobacterium, Humicoccus, Ilumatobacter, Salinibacterium, Aeribacillus, Caldalkalibacillus, Exiguobacterium, Lactobacillus, Lysinibacillus, Oceanobacillus, Pasteuria, Planomicrobium,
Solibacillus, and Trichococcus), that were abundant in
Krubera-Voronja cave, seem to be rare if any in other
caves.
Proteobacterial genera Escherichia, Halomonas,
and Pseudomonas were ubiquitous in all samples. These
three genera, along with the majority of the most

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Microbial diversity of Krubera-Voronja cave

numerous proteobacterial genera found in KruberaVoronja cave, were also detected in other caves (Barton et al. 2004; Bastian et al. 2009; Portillo et al. 2009;
Engel et al. 2013; Grin et al. 2014). We did not nd
any information about Albidiferax, Alkanindiges, Enhydrobacter, Haliea, Nitrosococcus, Pseudoxanthomonas,
and Rugamonas in the caves; it thus seems that these
genera have not been detected in the caves to date or
they were very sparse in these environments.
Some genera of the other phyla (Chryseobacterium,
Flavobacterium, and Nitrospira) that were among the
most abundant in Krubera-Voronja cave, have also been
identied in other caves (Carmichael et al. 2013; Grin
et al. 2014). As for the genera of Acidobacteria, Gemmatimonadetes, Planctomycetes, and Verrucomicrobia
that comprised 1% of all the sequences in some samples of Krubera-Voronja, we could not nd any information about their presence in other caves.
Some bacteria found in the caves (Apia, Bacillus, Inquilinus, Legionella, Pseudomonas, Staphylococcus, and Escherichia coli) can be pathogenic and cause
a danger for humans (Bastian et al. 2009; Jurado et al.
2010). Currently, pathogen presence in the environment
is assumed based on the detection of faecal indicator
bacteria (McLellan & Eren 2014). E. coli and enterococci were traditionally regarded as the faecal indicators, but the development of novel molecular techniques
allowed for other organisms, such as Bacteroidales, Bidobacterium and Lachnospiraceae, to be used as alternative faecal indicators. Our results demonstrated
that the genus Escherichia was among the most abundant genera in some samples from both rarely and
frequently visited locations. It is noteworthy that Escherichia did not comprise 1% of all the sequences from
samples 500WF1, 1410F2-F3, and 1640WF3 collected
in the underground camps, and the abundance of this
genus in them was expected. These results were in accordance with those for enterococci, Bidobacterium,
and Lachnospiraceae only 5 sequences were found
to belong to these taxa. Out of Bacteroidetes, the sequences of Flavobacteriales, Sphingobacteriales as well
as Cytophagales but not those of Bacteroidales dominated in Krubera-Voronja cave. Our results showed that
the faecal contamination is absent in Krubera-Voronja
cave.
Archaeal diversity of the deepest cave of the world
The studies of Archaea from the caves are scanty and
not exhaustive, although archaeal communities are suggested to play an important role in nutrient recycling within cave ecosystems (Kumaresan et al. 2014).
Usually, representatives of Euryarchaeota and Crenarchaeota are found in caves, although Korarchaeota,
Thaumarchaeota, and some candidate phyla were also
reported (Engel 2010; Carmichael et al. 2013; Jones
et al. 2014). A few 16S rRNA gene sequences of Crenarchaeota and Euryarchaeota as well as those of unclassied Archaea were detected in some samples from
Krubera-Voronja cave. It is known that low concentrations of DNA, partial fragmentation, and presence

1001
of enzymatic inhibitors in underground environments
complicate genetic analyses and interpretations (Epure
et al. 2014). While DNA fragmentation and PCR inhibitors in our DNA preparations can be excluded because of bacterial 16S rRNA genes were successfully
amplied, total DNA quantities were very low, and repeated DNA extraction as well as concentration of total DNA preparations were needed (data not shown).
Carmichael et al. (2013) showed that bacterial 16S
rRNA gene copies represented on average 95% of the
total estimated microbial (bacterial and archaeal) cell
numbers in the caves of Upper Tennessee River Basin,
while those of Archaea represented only 5%. We did
not perform the evaluation of the archaeal cell number
in Krubera-Voronja cave, but if the percentage is close
to that reported in literature, it could be the reason for
such extremely low number of archaeal sequences in the
samples from Krubera-Voronja cave.
The other problem we encountered was the nonspecicity of archaeal primer pair. A large percentage
of bacterial sequences in the data set, obtained from
barcoded pyrosequencing with archaeal primers, was
also reported previously (Porat et al. 2010). Our results
clearly showed that 16S rRNA gene sequences from a
range of bacterial phyla can be amplied using the S-DArch-0519-a-S-15/S-D-Arch-1048-a-A-17 primer pair.
As the percentage of archaeal sequences was higher
in the Archaea-rich microbial community (14.36% vs.
3% in Krubera-Voronja samples), we concluded that
both the primer non-specicity and possibly very low
archaeal DNA quantity in the samples from KruberaVoronja cave contributed to extremely low coverage of
archaeal 16S rRNA gene sequences in our data set.
Conclusions
In conclusion, our results demonstrated high bacterial
diversity at the phylum and genus level. We have denitely showed that the bacterial diversity at the phylum
level depends on both the sampling depth and the frequency of human visits in Krubera-Voronja cave. The
total number of bacterial genera both per phylum and
per sample correlated with the frequency of human visits but not with the sampling depth. Archaeal diversity
should be re-examined using quantitative PCR with
other Archaea-domain specic primers in order to establish the reasons for the extremely low coverage of
archaeal 16S rRNA gene sequences in our present data
set.

Acknowledgements
This work was supported by the Research Council of
Lithuania (grant No. MIP-005/2014) and the National Geographics Global Exploration Fund (grant No. GEFNE50Y12). Lithuanian caving club Aenigma and Ukrainian
Speleological Association are acknowledged for their cooperation in sampling. We would like to express our gratitude
to Vytautas Zurauskas for his valuable consultations and
assistance with bioinformatics.

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Authenticated
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Received April 20, 2015

Accepted August 18, 2015