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Down into the Earth: Microbial diversity of the deepest cave of the world

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DOI: 10.1515/biolog-2015-0127

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 Biologia 70/8: 989—1002, 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 different 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 first report on the microbial diversity in Krubera-Voronja cave.
Key words: pyrosequencing; Krubera-Voronja; speleomicrobiology; 16S rRNA gene; deep cave; microbial diversity.

Introduction bridization, denaturing gradient gel electrophoresis and

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 rela-
tively stable at or near the mean annual surface temper-
ature (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 at-
tract considerable attention of microbiologists in terms
of biodiversity and metabolism given the unique envi-
ronment.
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). Krubera-
Voronja 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 first 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 mi-
crobial diversity in the caves. Fluorescence in situ hy-
cloning of 16S rRNA gene were used for the cultivation-
independent 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 charac-
terize microbial diversity in Krubera-Voronja cave.
We hypothesized that this previously non-investigated
habitat contains unique communities of prokaryotes.
Cultivation-independent method, namely barcoded py-
rosequencing of 16S rRNA gene, was used for our anal-
ysis.
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, drink-
able water from the underground camps as well as coloured
spots from cave walls. Sampling sites were chosen consider-
ing the different depth and intensity of human activities. Mi-
crobiota from the rarely visited locations was considered to
be the indigenous microbiota of the cave and was compared

* Corresponding author

2015
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990 I. Kieraite-Aleksandrova et al.

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 con-
taining contaminants associated with human visits. The in-
digenous microbiota from the different depth was supposed
to differ because of the different supply of organic carbon
sources. In total, 26 samples were collected from Krubera-
Voronja cave. Fourteen samples were collected from the fre-
quently visited locations, and 12 samples – from rarely vis-
ited branches of the cave. Seven samples were from the up-
per part (220–230 m in depth), 2 samples – from the mid-
dle part (500–700 m in depth), and 17 samples – from the
lower part (1215–1640 m in depth) of the cave. All sam-
ples 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 amplification
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 se-
quence (TCAG), a multiplex identifier (a barcode) and the
domain-specific 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 identifier
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-specific reverse primer: 685R primer (5’-
TCT ACG CAT TTC ACC GCT AC-3’) for Bacteria and S-
D-Arch-1048-a-A-17 primer (5’-CGR CRG CCA TGY ACC
WC-3’) for Archaea (Klindworth et al. 2013). For Bacte-
ria, the expected PCR product size was 678 bp (excluding
454 Primers, the key sequences and the barcodes) and cov-
ered V1–V4 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 se-
quences and the barcodes) and covered V4-V6 variable re-
gions (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

Sample No. Material R/FVL Depth (m) T ( ◦C) H (%) DSPA

220F1 Soil (Crimea meander) Frequently 220 3 ∼ 69 Bact

220F2 Clay from ceiling (Crimea meander) Frequently 220 3 ∼ 69 Arch
220F3 Deposited carbide from carbide lamps Frequently 220 3 ∼ 69 Bact
(Crimea meander)
220F4 Green clay (Crimea meander) Frequently 220 3 ∼ 69 Bact
230R1 White calcite crystals (non- Rarely 230 3 ∼ 76 Bact
Kujbyshevskaja meander)
230R2 Soil (non-Kujbyshevskaja meander) Rarely 230 3 ∼ 76 Bact
230R3 Helictites (non-Kujbyshevskaja Rarely 230 3 ∼ 76 Arch
meander)
500WF1 Water Frequently (underground camp) 500 3 >90 Bact
700WF1 Water Frequently (underground camp) 700 4 >90 Bact
1215WF1 Water Frequently (underground camp) 1215 6 >90 Bact
1215F2 Soil Frequently (underground camp) 1215 6 >90 Arch
1350R1 Black soil Rarely visited branch 1350 6 >90 Arch, Bact
1350R2 White clay Rarely visited branch 1350 6 >90 Arch, Bact
1350R3 Yellow clay Rarely visited branch 1350 6 >90 Arch, Bact
1410WF1 Water Frequently (underground camp) 1410 8 >90 Bact
1410F2 Soil, rust Frequently (underground camp) 1410 8 >90 Arch, Bact
1410F3 Black soil Frequently (underground camp) 1410 8 >90 Arch, Bact
1500R1 Red spots Rarely 1500 6 >90 Arch, Bact
1530R1 White calcite crystals Rarely 1530 6 >90 Bact
1550R1 Irregularly-shaped spots Rarely visited ledge 1550 6 >90 Bact
1550R2 Soil Rarely visited ledge 1550 6 >90 Bact
1550R3 Soil Rarely visited ledge 1550 6 >90 Bact
1620R1 Yellow and red spots Rarely 1620 6 >90 Bact
1640F1 Soil Frequently (underground camp) 1640 6 >90 Arch
1640F2 Soil Frequently (underground camp) 1640 6 >90 Arch, Bact
1640WF3 Water Frequently (underground camp) 1640 6 >90 Bact

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

Bact, Bacteria-specific primers.

Total DNA extraction and amplification of 16S rRNA gene
Total DNA from the samples was extracted using the in-
nuSPEED Soil DNA kit (Analytik Jena AG). For ampli-
fication, total DNA from samples 1550R1 and 1550R3 as
well as 1350R1, 1350R2, and 1350R3 (Table 1) was pooled.
Bacterial 16S rRNA genes were amplified in 50 µL of reac-
tion mixture containing the DreamTaq Green PCR Master
Mix (2X) (Thermo Fisher Scientific), 0.25 µM each primer,
and 10 ng of total DNA. Amplifications of bacterial 16S
rRNA gene were conducted under the following conditions:
initial denaturation at 95 ◦C for 2 min followed by 29 cy-
cles each consisting of 95 ◦C for 1 min, 50 ◦C for 2 min and
72 ◦C for 3 min with a final extension step at 72 ◦C for 7 min
in an Eppendorf thermal cycler. Archaeal 16S rRNA genes
were amplified in 50 µL of reaction mixture containing PCR
buffer with (NH4 )2 SO4 , 2 mM MgCl2 , 0.2 mM each dNTP,
0.25 mM each primer, 1.25 U recombinant TaqDNA Poly-
merase (Thermo Fisher Scientific) and 10 ng of total DNA.
Amplifications 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 final exten-
sion step at 72 ◦C for 7 min in an Eppendorf thermal cycler.
Products of amplifications were analysed by electrophoresis
through 1% agarose gel. PCR products were purified using
the GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
Sequence analysis
Purified PCR products were subjected for pyrosequencing.
Barcoded pyrosequencing of bacterial 16S rRNA gene was
performed by the LGC Genomics GmbH (Germany) us-
ing a GS FLX Titanium platform (Roche). Barcoded py-
rosequencing 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 MultiClasifier 1.1. at the 80%
confidence threshold (Cole et al. 2014). Data were normal-
ized 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
differences in the bacterial communities from the different
groups of samples. ANOSIM R values (strength of dissimi-
larity) of R = 0 indicate a high degree of community sim-
ilarity, 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 significant at P < 0.05.
BioProject accession number
Pyrosequencing data were deposited in the GenBank Se-
quence 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 specific primers, and 5821 sequences

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992
Fig. 2. Relative bacterial diversity at the phylum level in Krubera-
Voronja 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 specific 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, 97.36–99.54% of all the se-
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I. Kieraite-Aleksandrova et al.
quences could be assigned to the phylum irrespective
of the sampling depth as well as the frequency of hu-
man visits. For six samples (230R1, 500WF1, 1350R1-
R3, 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 identified as representatives of a
particular phylum.
In total, 24 phyla were identified in the samples
from Krubera-Voronja cave. The dominant phyla were
Proteobacteria and Actinobacteria followed by Firmi-
cutes, Bacteroidetes and Acidobacteria (Fig. 2). The
percentage of other phyla (Armatimonadetes, Chlamy-
diae, Chlorobi, Chloroflexi, Cyanobacteria, Deinococ-
cus-Thermus, Elusimicrobia, Fusobacteria, Gemmati-
monadetes, Nitrospirae, Planctomycetes, Spirochaetes,
Verrucomicrobia, OD1, OP11, WS3, BRC1, SR1, and
TM7) was below 1%. The unclassified Bacteria consti-
tuted 6% of all the sequences. Not a single sequence of
Archaea was detected using Bacteria-domain specific
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 identified as representatives of a
particular genus. For most samples from the frequently
visited locations of the cave 86.2–95.0% of all the se-
quences 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%, respec-
tively, of all the sequences in these samples identified
as the sequences of the particular genera.
Bacterial diversity at the rarely visited locations of
Krubera-Voronja cave
In total, 16S rRNA gene sequences of 20 phyla were
identified in the samples from the rarely visited loca-
tions. Combined data from all the samples from the
rarely visited locations of the cave showed the domi-
nant position of Proteobacteria followed by Actinobac-
teria, Firmicutes, unclassified Bacteria, and Acidobac-
teria (Fig. 2). Bacteroidetes constituted 2% of all the
sequences similar as did the sequences from the remain-
ing phyla. The sequences of Proteobacteria, Actinobac-
teria, Firmicutes, Acidobacteria and Cyanobacteria as
well as those of unclassified Bacteria were detected
in all samples. Bacteroidetes were also ubiquitous, ex-
cept for samples 1530R1 and 1550R1+R3. Other phyla
(Armatimonadetes, Chlorobi, Chloroflexi, Deinococcus-
Thermus, Elusimicrobia, Gemmatimonadetes, Nitrospi-
rae, Planctomycetes, Spirochaetes, Verrucomicrobia, as
well as candidate phyla WS3, OP11 and OD1) were
identified in 1–5 samples from the rarely visited loca-
tions of the cave, from 0.02% to 3.14% of all the se-
quences per phylum in different samples.
Because of the differences in temperature, humid-
ity as well as the supply of organic carbon in the upper
and lower parts of the cave, we chose to compare py-
Microbial diversity of Krubera-Voronja cave 993

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 (1350–1620 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 different 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 31 1 5 1 1 1 21 1 1 2 56 2 1 1
230R2 3 29 8 1 1 28 47
LPC, S
1350R1-R3 15 31 3 2 1 4 1 1 1 19 1 1 4 26 2 1 1
1500R1 7 17 9 1 1 17 1 1 48 2 1
1530R1 2 11 1 11 1 14
1550R1+R3 1 18 2 16 10
1550R2 1 19 1 1 13 14
1620R1 13 27 1 12 3 1 1 18 1 1 4 74 1 5 1 1 1
FVL, UPC, S
220F1 11 20 3 16 4 2 1 35 1 1 2 56 2 1 1
220F3 1 26 3 1 1 28 33
220F4 7 13 6 3 1 17 1 1 31 1 1
MPC, W
500WF1 13 22 3 13 1 7 1 1 23 2 1 1 6 70 2 4 1 1 1 1
700WF1 2 21 8 1 1 21 1 1 57
LPC, S
1410F2 9 59 4 27 7 1 2 36 1 1 8 94 7 1 1 1
1410F3 8 21 2 10 4 1 8 1 1 7 44 5 1 1
1640F2 5 15 1 9 1 10 1 41 1 1
LPC, W
1215WF1 3 13 6 1 1 1 20 1 1 48 1 1 1
1410WF1 9 17 3 11 1 1 2 25 1 1 1 3 45 1 1
1640WF3 14 38 2 25 5 1 1 34 1 1 3 101 5 1 1 1 1
a 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, Chloroflexi; 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 (230R1-
R2) 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 significantly differed 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, unclassified 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 abun-
dant. 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 up-
per one. In contrast with the upper part, Chloroflexi,
Nitrospirae and Planctomycetes each constituted 1%

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994 I. Kieraite-Aleksandrova et al.
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

UPC, S

230R1 135 Microbacterium (42.64), Pseudomonas (7.42), Es- Staphylococcus (3.94), Gp3 (3.62; Acidobacteria)
Micrococcus (2.69), cherichia (2.74), Acineto- Planomicrobium (1.83),
Corynebacterium (1.64), bacter (1.98) Geobacillus (1.71), Strep-
Propionibacterium (1.26) tococcus (1.21)

230R2 117 Propionibacterium Halomonas (14.40), Aeribacillus (7.0), Staphy-

(21.44), Micrococcus Herbaspirillum (11.42), lococcus (2.75), Caldal-
(3.91), Nesterenkonia Escherichia (8.81), kalibacillus (2.02), Lacto-
(3.35), Corynebacterium Acinetobacter (2.31) bacillus (2.02), Bacillus
(2.80) (1.11)

LPC, S

1350R1-R3 114 Arthrobacter (3.32),
Nesterenkonia (1.96),
Propionibacterium (1.42)
Halomonas (4.15), Aeribacillus (3.32), Bacil- Gp6 (4.27; Acidobacte-
Psychrobacter (2.85), lus (2.31), Caldalkalibacil- ria), Nitrospira (2.90; Ni-
Herbaspirillum (1.90), lus (1.42) trospirae), WS3 genera-
Escherichia (1.54), incertae sedis (1.96;
Albidiferax (1.36) WS3), Gp4 (1.78; Aci-
dobacteria), Gp16 (1.66;
Acidobacteria), Gp17
(1.42; Acidobacteria),
Gemmatimonas (1.30;
Gemmatimonadetes)

1500R1 105 Pseudomonas (65.80), Staphylococcus (2.31), Flavobacterium (3.33;

Acinetobacter (6.70), Aerococcus (1.77) Bacteroidetes)
Perlucidibaca (1.91)

1530R1 40 Nesterenkonia (13.65), Herbaspirillum (17.71), Caldalkalibacillus (8.12),

Propionibacterium (4.80), Halomonas (13.65), Aeribacillus (4.06),
Arthrobacter (2.95), Acinetobacter (9.23), Staphylococcus (1.48)
Corynebacterium (1.48), Pseudomonas (1.48),
Pseudonocardia (1.11), Psychrobacter (1.11)
Rothia (1.11)

1550R1+R3 47 Propionibacterium Psychrobacter (20.45), Lysinibacillus (2.99),

(10.72), Arthrobacter Halomonas (4.99), Solibacillus (2.24), Paeni-
(8.48), Micrococcus Herbaspirillum (3.24), bacillus (2.0), Bacillus
(7.98), Corynebacterium Acinetobacter (1.99), (1.75), Staphylococcus
(3.99), Brachybacterium Enhydrobacter (1.50) (1.50), Planococcaceae-
(1.75), Nesterenkonia incertae sedis (1.25)
(1.20)

1550R2 49 Propionibacterium Halomonas (6.77), Aeribacillus (6.13),

(18.39), Micrococcus Herbaspirillum (3.22), Geobacillus (3.55), Bacil-
(10.32), Corynebac- Psychrobacter (1.61), lus (2.90), Caldalkalibacil-
terium (6.45), Nesterenko- Acinetobacter (1.29) lus (1.94), Lysinibacil-
nia (4.84), Arthrobacter lus (1.94), Lactobacillus
(3.23), Brachybacterium (1.61), Staphylococcus
(2.58) (1.29)

1620R1 165 Arthrobacter (3.71), Pseudomonas (32.18), Flavobacterium

Pseudonocardia (1.14)
Escherichia (11.20), Al- (2.13; Bacteroidetes),
bidiferax (3.55), Acine- Ohtaekwangia (1.42;
tobacter (2.76), Hy- Bacteroidetes)
drogenophaga (2.21), Ni-
trosospira (1.18), Pseu-
doxanthomonas (1.18),
Massilia (1.14)

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 com- Chlorobi, Elusimicrobia, Spirochaetes, and candidate
prised further 2% in the lower part of the cave. Phyla phylum OD1 were not detected in the upper part of the

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Microbial diversity of Krubera-Voronja cave 995

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 (500–700 m in depth) and lower (1215–1640 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 identified in the samples from the lower
part.
The number of genera per phylum ranged from 1
to 74 in different samples from the rarely visited loca-
tions (Table 2). The highest number of genera was iden-
tified 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 (Ta-
ble 3). The genera of Bacteroidetes were also identi-
fied in samples 1500R1 and 1620R1, and those of Aci-
dobacteria were found in samples 230R1 and 1350R1-
R3. 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 fre-
quently visited locations of the cave showed the dom-
inant position of Proteobacteria (42%) (Fig. 2). The
16S rRNA genes of Actinobacteria, Firmicutes, Pro-
teobacteria, Acidobacteria and Bacteroidetes as well as
those of unclassified Bacteria were detected in all sam-
ples. Chloroflexi and Cyanobacteria were also ubiqui-
tous, except for sample 220F3 (without Chloroflexi) and
sample 1640F2 (without Cyanobacteria). The minor

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996
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, Chlamy-
diae, Deinococcus-Thermus, Fusobacteria, Gemmati-
monadetes, Nitrospirae, Planctomycetes, Spirochaetes,
Verrucomicrobia, as well as candidate phyla WS3,
OP11, OD1, BRC1, SR1 and TM7 were identified 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 different
samples.
ANOSIM showed that the bacterial communities
from the upper (220 m), middle (500–700 m) and lower
(1215–1640 m) parts of the cave were significantly dif-
ferent (Rupper−middle = 0.332, Rupper−lower = 0.291,
Rmiddle−lower = 0.316; P < 0.05). The phylum Pro-
teobacteria was the dominant phylum (33–44% of all
the sequences) in the frequently visited places irrespec-
tive of the sampling depth (Fig. 4). While Actinobac-
teria followed Proteobacteria in the samples from both
the upper and the lower parts of the cave, phylum Fir-
micutes 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). Unclassified Bacteria comprised 11–14%
in the upper and middle parts, while they constituted
only 3% in the lower part of the cave. The percentage
of the sequences of Chloroflexi 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, Fu-
sobacteria, and Spirochaetes were not found in this part
of the cave, while were identified 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
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I. Kieraite-Aleksandrova et al.
of the cave, while they were identified in the middle
and lower parts, respectively. The interesting trend was
observed for Bacteroidetes – the percentage of their se-
quences 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 1215–1640 m (Fig. 4).
As the samples of both water and sediments were
collected in the frequently visited places, the analy-
sis ‘water vs. sediments’ was carried out. Our analy-
sis showed that these two bacterial communities were
significantly different (ANOSIM R = 0.536; P < 0.05).
The main difference between both types of the samples
was determined to be the percentage of the sequences of
Actinobacteria and Proteobacteria (Fig. 5). The domi-
nant 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 Bac-
teroidetes, namely Bacteroidales, are known to be ex-
cellent 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 1–3
sequences per sample if any. Flavobacteriales was the
prevalent order of Bacteroidetes (data not shown), and
Flavobacterium sequences were among the most numer-
ous genera in the frequently visited regions of the cave
(Table 4).
The number of genera per phylum was from 1 to
101 in the different samples from the frequently vis-
ited locations (Table 2). The highest number of genera
was identified for Proteobacteria in all samples. The
total number of genera varied from 82 to 259 genera
Microbial diversity of Krubera-Voronja cave 997
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

UPC, S

220F1 156 Microbacterium (23.58), Massilia (6.23), Pseu- Streptococcus (3.50), Gp3 (4.88; Acidobacteria),
Propionibacterium (2.55), domonas (4.85), Aeribacillus (2.60), Sediminibacterium (1.83;
Corynebacterium (2.03), Halomonas (4.28), Staphylococcus (2.35), Bacteroidetes)
Micrococcus (1.80) Stenotrophomonas (3.18), Geobacillus (1.25)
Escherichia (3.03), Acine-
tobacter (2.65), Sphin-
gomonas (1.08)

220F3 93 Propionibacterium (8.92), Halomonas (20.66), Aeribacillus (11.52), Cal-

Nesterenkonia (5.74), Herbaspirillum (11.38), dalkalibacillus (4.23),
Corynebacterium (3.28), Escherichia (2.73), Acine- Bacillus (2.05), Staphy-
Micrococcus (3.14), tobacter (1.59), Psy- lococcus (1.96), Geobacil-
Arthrobacter (1.59) chrobacter (1.18) lus (1.37), Oceanobacil-
lus (1.23), Lactobacillus
(1.18)

220F4 82 Propionibacterium (1.43), Pseudomonas (8.74), Planomicrobium (1.72),

Corynebacterium (1.10), Acinetobacter (1.76), Exiguobacterium (1.05)
Micrococcus (1.0) Halomonas (1.43)

MPC, W

500WF1 174 Pseudomonas (17.18), Al- Planomicrobium (13.59), Gp6 (2.93; Acidobac-
bidiferax (2.66), Paracoc- Exiguobacterium (7.04), teria), WS3 genera-
cus (1.26) Staphylococcus (2.39) incertae sedis (1.29; WS3),
Flavobacterium (4.70;
Bacteroidetes)

700WF1 113 Propionibacterium (2.45), Escherichia (34.26), Pseu- Staphylococcus (2.45),

Corynebacterium (1.71), domonas (6.27), Acine- Streptococcus (1.14)
Micrococcus (1.43) tobacter (4.50), Massilia
(3.25), Citrobacter (3.14),
Alkanindiges (1.08)

LPC, S

1410F2 259 Arthrobacter (30.88), Hu- Albidiferax (3.75), Pseu- Trichococcus (4.76) Flavobacterium (3.41;
micoccus (2.01), Salin- doxanthomonas (2.41), Bacteroidetes), Gp4 (2.69;
ibacterium (1.87), Pro- Herbaspirillum (1.88), Acidobacteria)
pionibacterium (1.62), Polaromonas (1.64),
Cryobacterium (1.0) Halomonas (1.61), Psy-
chrobacter (1.36), Acine-
tobacter (1.28), Pseu-
domonas (1.15)

1410F3 114 Ilumatobacter (2.77) Pseudomonas (8.20), Pasteuria (1.57) Ferruginibacter (18.05;
Haliea (6.31), Rhizobacter Bacteroidetes), Ter-
(2.37), Halomonas (1.13) rimonas (3.05; Bac-
teroidetes), Gp6 (4.26;
Acidobacteria), Haloferula
(1.93; Verrucomicrobia),
Gemmata (1.45; Planc-
tomycetes), Gp4 (1.29;
Acidobacteria)

1640F2 85 Ilumatobacter (4.64) Acinetobacter (11.59), OP11 genera-incertae

Aquicella (7.32), Pseu- sedis (1.23; OP11)
domonas (3.48), Ni-
trosococcus (2.32), En-
hydrobacter (1.45),
Escherichia (1.30),
Halomonas (1.23), Pseu-
doxanthomonas (1.16),
Massilia (1.16), Legionella
(1.09)

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998 I. Kieraite-Aleksandrova et al.
Table 4. (continued)

Samples TNG Actinobacteria Proteobacteria Firmicutes Other

1215WF1 98 Pseudomonas (59.08), Es- Flavobacterium (1.18;

cherichia (16.81), Acineto- Bacteroidetes)
bacter (2.72), Citrobacter
(1.4)

1410WF1 122 Propionibacterium (8.49), Acinetobacter (13.20),
Micrococcus (3.70), Halomonas (5.80), Pseu-
Corynebacterium (2.86), domonas (5.63), Es-
Nesterenkonia (1.01) cherichia (3.28), Mas-
silia (2.69), Rheinheimera
(2.10), Stenotrophomonas
(2.10), Cellvibrio (1.26),
Enhydrobacter (1.10)
Aeribacillus (2.52), Strep-
tococcus (1.60), Staphylo-
coccus (1.60)
Chryseobacterium
(6.73; Bacteroidetes),
Wautersiella (2.35;
Bacteroidetes),
Flavobacterium (1.68;
Bacteroidetes)

1640WF3 235 Propionibacterium (6.68), Albidiferax (4.86), Flavobacterium (10.18;

Arthrobacter (6.17) Acinetobacter (3.40), Bacteroidetes)
Dechloromonas (2.80),
Alkanindiges (2.0), Rug-
amonas (1.32), Po-
laromonas (1.28), Massilia
(1.24), Halomonas (1.08)

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, Proteobac-
teria 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 se-
quences per sample) were identified in all the samples
except for 220F3-F4, 700WF1, and 1640F2. The gen-
era of Acidobacteria, Verrucomicrobia, Planctomycetes,
OP11, and WS3 were also identified in some samples
(Table 4). The abundance of some genera depending
on the sampling depth is noteworthy. For example, Mi-
crobacterium 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 different types of samples
The influence 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 fre-
quency of human visits were disregarded in this anal-
ysis. ANOSIM showed that the bacterial communities
from these three types of the samples differed signif-
icantly (Rsoil−speleothems = 0.485, Rsoil−spots = 0.622,
Rspeleothems−spots = 0.591; P < 0.05). Acidobacteria,
Actinobacteria, Bacteroidetes, Firmicutes, Proteobac-
teria as well as unclassified Bacteria were detected in
all three types of samples. Planctomycetes and Chlo-
roflexi 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 difference 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 con-
trast, phylum Proteobacteria was the absolute leader
in the latter samples – 79% of all sequences. The per-
centage of Firmicutes was similar in soil samples and
speleothems (11 and 13%, respectively), but this phy-
lum 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-specific primers (Ta-
ble 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 unclassified Archaea from sample
1640F1. The larger set of archaeal sequences (24 se-
quences) was obtained only from samples 1350R1-R3.
Out of these 24 sequences, 15 sequences were found to
belong to unclassified Archaea, 7 sequences to Crenar-
chaeota, and 2 sequences to Euryarchaeota. All crenar-
chaeal 16S rRNA gene sequences were identified as un-
classified Thermoprotei. Euryarchaeal 16S rRNA gene
sequences were identified as unclassified Euryarchaeota.
At the initial step of our work we used Archaea-
domain-specific primer pair S-D-Arch-0519-a-S-15/S-
DArch-1041-a-A-18 recommended by Klindworth et al.

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Microbial diversity of Krubera-Voronja cave
Fig. 6. Comparison of bacterial diversity at the phylum level in
the samples of the different nature. Only those phyla that con-
stituted ≥1% of all the sequences in the combined data sets are
shown.
(2013) as the most suitable primer pair for sequenc-
ing technologies like Roche’s 454. Unfortunately, we
could not obtain PCR product of the correct size
using this primer pair. Therefore, we chose the S-
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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 unspe-
cific. 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 se-
quences of phyla Actinobacteria, Firmicutes (Bacilli,
Clostridia and Erysipelotrichia), Proteobacteria (α-
and γ-Proteobacteria) and Tenericutes constituted the
majority of bacterial sequences, but 16S rRNA genes of
Acidobacteria, Armatimonadetes, Bacteroidetes, Chlo-
roflexi, Cyanobacteria, Planctomycetes, Spirochaetes,
Synergistetes, Thermotogae and Verrucomicrobia were
also detected. Such baffling results impelled us to
verify the S-D-Arch-0519-a-S-15/S-D-Arch-1048-a-A-
17 primer pair using Archaea-rich community from the
anaerobic digester. In total, 6615 sequences were ob-
tained, but methanogenic Euryarchaeota constituted
only 14.36%. Other sequences belonged to those of
Firmicutes (68.86%), unclassified Bacteria (15.93%),
Tenericutes (0.47%), and Actinobacteria (0.21%); a few
sequences were also obtained for Bacteroidetes, Pro-
teobacteria, Spirochaetes, Synergistetes and Thermo-
togae.
Discussion
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 iden-
tified in caves (Engel 2010). Not all of these phyla
were detected in the particular cave, i.e. different num-
bers of bacterial phyla were detected in different 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 nu-
merous 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 Krubera-
Voronja 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-
1000
tions, i.e. the indigenous microbiota of the cave, signifi-
cantly differed from that of the frequently visited loca-
tions (R = 0.315; P < 0.05). For the fifth phylum, Bac-
teroidetes, and for the minor phyla Chloroflexi, Planc-
tomycetes, and Verrucomicrobia, the other trend was
observed – the larger percentage of these phyla was es-
tablished 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 dif-
ferences at the phylum level diversity between these
two types of the samples were associated with the pres-
ence/absence of the minor phyla Chlamydiae, Elusimi-
crobia, Fusobacteria, BRC1, SR1, and TM7.
As only microbiomes of a quite shallow caves were
studied untill now, it was impossible to investigate the
differences in the bacterial diversity depending on the
sampling depth. But the depth of Krubera-Voronja cave
allowed us to do this research. Our results definitely
showed that bacterial diversity at the phylum level de-
pended on the sampling depth. The change of the first
prevalent phylum in the samples from the rarely visited
locations was the most surprising. While phylum Pro-
teobacteria was the most abundant in the lower parts
of the cave, Actinobacteria became the first 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 dif-
ferences in the organic carbon supply in the upper and
lower parts of the cave: Actinobacteria are typical het-
erotrophs 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 first
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 different 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 Al-
tamira cave. In contrast, Cuezva et al. (2012) reported
that the grey spots collected from the walls and ceil-
ing of the same Altamira cave were composed of Acti-
nobacteria and calcium carbonate crystals. Percentage
of Firmicutes in the samples collected from the coloured
spots in Krubera-Voronja cave was low, and these re-
sults 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 cave’s speleothems.
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I. Kieraite-Aleksandrova et al.
It should be noted that phyla Acidobacteria, Actinobac-
teria, and Proteobacteria were revealed to dominate
in bacterial communities in the different samples from
speleothems of Kartchner Caverns (Ortiz et al. 2013).
In accordance with the phylum level diversity, the
most numerous genera belonged mainly to Actinobac-
teria, Proteobacteria and Firmicutes, irrespective of the
frequency of human visits. The total number of the gen-
era both per phylum and per sample correlated with
the frequency of human visits – it was higher in the fre-
quently visited locations than in the rarely visited ones.
Our findings that the frequent visits resulted in higher
diversity at both phylum and genus level in Krubera-
Voronja cave are in contrast with the data reported
previously, when it was shown that the microbial diver-
sity 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 unclassified 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 abun-
dance of some genera to a certain degree depended on it.
For example, although the genus Microbacterium was
identified 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 identified
in the samples from the different sampling depth (data
not shown), Enhydrobacter, Ilumatobacter, and Pseu-
doxanthomonas were numerous only in the lower part
of the cave albeit not in all the samples.
Some actinobacterial genera (Nocardia, Mycobac-
terium, Gordonia, Rhodococcus, and Streptomyces)
were reported to be widely spread in the caves (Ju-
rado et al. 2010), but these genera were not among
the most numerous in Krubera-Voronja cave. Instead of
them, actinobacterial genera Arthrobacter, Corynebac-
terium, Micrococcus, Nesterenkonia, and Propionibac-
terium were ubiquitous in all samples as well as
among the most numerous genera in the majority
of the samples. These genera as well as Brachybac-
terium, Microbacterium, Pseudonocardia, and Rothia
were previously also detected in other caves (Groth
et al. 2001; Portillo et al. 2009; Griffin et al. 2014).
Out of Firmicutes, only Staphylococcus was found in
all the samples. This genus as well as Aerococcus,
Bacillus, Geobacillus, Paenibacillus, and Streptococ-
cus were also detected in other caves (Groth et al.
2001; Engel 2010; Griffin et al. 2014). Other genera
of Gram-positive bacteria (Cryobacterium, Humicoc-
cus, Ilumatobacter, Salinibacterium, Aeribacillus, Cal-
dalkalibacillus, Exiguobacterium, Lactobacillus, Lysini-
bacillus, 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
Microbial diversity of Krubera-Voronja cave
numerous proteobacterial genera found in Krubera-
Voronja cave, were also detected in other caves (Bar-
ton et al. 2004; Bastian et al. 2009; Portillo et al. 2009;
Engel et al. 2013; Griffin et al. 2014). We did not find
any information about Albidiferax, Alkanindiges, Enhy-
drobacter, 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
identified in other caves (Carmichael et al. 2013; Griffin
et al. 2014). As for the genera of Acidobacteria, Gem-
matimonadetes, Planctomycetes, and Verrucomicrobia
that comprised ≥1% of all the sequences in some sam-
ples of Krubera-Voronja, we could not find any infor-
mation about their presence in other caves.
Some bacteria found in the caves (Afipia, Bacil-
lus, Inquilinus, Legionella, Pseudomonas, Staphylococ-
cus, 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 entero-
cocci were traditionally regarded as the faecal indica-
tors, but the development of novel molecular techniques
allowed for other organisms, such as Bacteroidales, Bi-
fidobacterium and Lachnospiraceae, to be used as al-
ternative faecal indicators. Our results demonstrated
that the genus Escherichia was among the most abun-
dant genera in some samples from both rarely and
frequently visited locations. It is noteworthy that Es-
cherichia 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 ac-
cordance with those for enterococci, Bifidobacterium,
and Lachnospiraceae – only 5 sequences were found
to belong to these taxa. Out of Bacteroidetes, the se-
quences of Flavobacteriales, Sphingobacteriales as well
as Cytophagales but not those of Bacteroidales domi-
nated 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 sug-
gested to play an important role in nutrient recy-
cling within cave ecosystems (Kumaresan et al. 2014).
Usually, representatives of Euryarchaeota and Crenar-
chaeota 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 Cre-
narchaeota and Euryarchaeota as well as those of un-
classified Archaea were detected in some samples from
Krubera-Voronja cave. It is known that low concen-
trations of DNA, partial fragmentation, and presence
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1001
of enzymatic inhibitors in underground environments
complicate genetic analyses and interpretations (Epure
et al. 2014). While DNA fragmentation and PCR in-
hibitors in our DNA preparations can be excluded be-
cause of bacterial 16S rRNA genes were successfully
amplified, total DNA quantities were very low, and re-
peated DNA extraction as well as concentration of to-
tal 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 non-
specificity 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 amplified using the S-D-
Arch-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-specificity and possibly very low
archaeal DNA quantity in the samples from Krubera-
Voronja 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 defi-
nitely showed that the bacterial diversity at the phylum
level depends on both the sampling depth and the fre-
quency 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 vis-
its but not with the sampling depth. Archaeal diversity
should be re-examined using quantitative PCR with
other Archaea-domain specific primers in order to es-
tablish 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 Ge-
ographic’s Global Exploration Fund (grant No. GEFNE50-
Y12). Lithuanian caving club ‘Aenigma’ and Ukrainian
Speleological Association are acknowledged for their coop-
eration in sampling. We would like to express our gratitude
to Vytautas Zurauskas for his valuable consultations and
assistance with bioinformatics.
1002
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Received April 20, 2015
Accepted August 18, 2015