BACTERIAL INFLUENCE ON SPELEOTHEM OXYGEN ISOTOPE COMPOSITION 199
GEOLOGICA CARPATHICA, 54, 3, BRATISLAVA, JUNE 2003
199204
BACTERIAL INFLUENCE ON SPELEOTHEM OXYGEN ISOTOPE
COMPOSITION: AN EXAMPLE BASED ON CAVE PISOIDS
FROM PERLOVÁ CAVE (SLOVAKIA)
MICHA£ GRADZIÑSKI
Institute of Geological Sciences, Jagiellonian University, Oleandry Str. 2a, 30-063 Kraków, Poland; gradzinm@ing.uj.edu.pl
(Manuscript received April 4, 2002; accepted in revised form December 12, 2002)
Abstract: The origin of recently growing irregular cave pisoids in Perlová Cave (Ve¾ká Fatra Mts, Slovakia) seems to be
due to the activity of hydrogen-oxidizing bacteria. Several samples of water and freshly deposited calcite from cave
pisoids were analysed for stable oxygen isotope ratios. The obtained values were checked using the ONeil equation.
Almost all the calcite samples display values more positive than their calculated counterparts. This phenomenon is
ascribed to a fractionation process mediated by bacteria. The light isotope of oxygen is preferentially taken up by the
hydrogen-oxidizing bacteria. It causes the relative enrichment in the heavy isotope in the bacterias surroundings, which
is recorded in the calcite precipitated around the bacterial cells.
Key words: Ve¾ká Fatra Mts, stable oxygen isotopes, bacterial influence, speleothems.
Introduction
Carbonate speleothems which grow under conditions of isoto-
pic equilibrium between calcite and ambient water are often
used as a tool in paleoclimatic reconstruction (cf. Schwarcz
1986; Gascoyne 1992). In contrast, much less attention has
been paid to the other speleothems of specific isotope compo-
sitions, since these are considered useless for paleo-
environmental studies.
The causes of disequilibrium in the C and O isotopes during
the growth of speleothems were usually attributed to the rapid
diffusion of CO
2
and/or to the evaporation effect (Hendy
1971). However, these processes of stable isotope fractionat-
ion have been only sporadically discussed so far. Study of re-
cent cave pisoids from Perlová Cave in Slovakia offers the
possibility of analysing and explaining a process of the stable
oxygen isotope fractionation between ambient water and
growing calcite speleothems.
Environmental setting
Perlová Cave (Perlová jaskyòa) lies in the Belanská Valley
in the northern part of the Ve¾ká Fatra Mountains (Great Fatra)
in Slovakia (Fig. 1) (Mrázik 1987). Its entrance is situated at
910 m a.s.l. The average temperatures in the region range from
4 °C to 7 °C, depending on altitude (Droppa 1975).
Perlová Cave is developed in Guttenstein bedded limestone
(Middle Triassic) of the Kríná Unit overthrusted over Meso-
zoic autochthonous cover of the crystalline core of the Ve¾ká
Fatra Mountains (Mahe¾ 1968). The 408 m-long cave is rich in
speleothems (Mrázik 1987; Holúbek & Kleskeò 1993). The
internal temperature ranges between 5.16.8 °C. The cave is
devoid of any permanent or ephemeral watercourses. Water is
pilled up only in small stepped rimstone pools (Fig. 2) located
in two places: in the Pearls Passage (Perlová chodba) and in
the Parliament Chamber (Parlament; Mrázik 1987). The depth
of the pools ranges from 2 cm to 6 cm and the size of the big-
gest is 1´1.2 m. The water is of HCO
3
CaMg type not dif-
fering from typical karst water in composition. Total dissolved
solids (TDS) fluctuates between 302 and 432 mg/dm
3
. The
saturation index (SI) indicates supersaturation both with re-
spect to the aragonite and calcite (Gradziñski 2001).
Individual pools host a dozen to several hundred pisoids of
various sizes. Irregular rough surfaces, irregular subtle lamina-
Fig. 1. Location of Perlová Cave.
200 GRADZIÑSKI
tion and lack of a nucleus characterizes the pisoids (Fig. 3).
Low-Mg calcite is the only autochthonous carbonate phase
that forms pisoids. The magnesium content in calcite does not
exceed 5600 ppm (i.e., 1.47 mole % of MgCO
3
). The pisoids
grow due to physiological processes generated by chemo-
lithoautotrophic hydrogen-oxidizing bacteria. The bacteria
grow in the mucilaginous biofilm that occurs on the surface of
the pisoids (Fig. 4). Hydrogen-oxidizing bacteria actively up-
take CO
2
from their surroundings (Aragno & Schlegel 1992)
resulting in a disequilibrium state of the solution and, conse-
quently, in precipitation of calcite upon the surface of pisoids.
Thus, the growth of the pisoids is controlled by bacterial meta-
bolic activity (Gradziñski 2001).
Materials and methods
Six rimstone pools with pisoids were selected for isotopic
examination. Three of these, P1, P5 and P6, were located in
the Pearls Passage and the other three, P8, P9 and P10, came
from the Parliament Chamber. Sampling of water for analysis
of isotopic composition in selected rimstone pools was car-
ried out twice: on November 11, 1998 and on May 19, 1999.
Eleven pisoids were selected for the analysis of stable isotope
composition. The diameters of the pisoids ranged from 1.2 to
2.5 cm.
Stable isotope ratios were analysed for the samples of water
and pisoids. The analyses of the water samples were conducted
in the Mass Spectrometry Laboratory of the Department of En-
vironmental Physics, the Faculty of Physics and Nuclear Engi-
neering, Academy of Mining and Metallurgy, Kraków. The
procedure used was a standard procedure for the equilibration
of a sample with gaseous CO
2
(
δ
18
O
w
) and reduction of water
on metallic uranium at a temperature of 650 °C (
δ
D). Oxygen
and hydrogen isotope ratios were measured using a FINNI-
GAN Delta S mass spectrometer. The analytical error for sin-
gle measurements is ±0.1 for
δ
18
O
w
and ±1 for
δ
D. Sta-
ble isotope ratios in water are presented here vs. SMOW
standard (cf. Hoefs 1997).
To define stable carbon and oxygen isotope ratios in the
pisoids, samples of ca. 10 mg weight were taken with Dremel
drilling machine. Where possible, the samples were taken from
isochronous horizons. The measurements of carbonates
δ
13
C
and
δ
18
O
c
were conducted with a mass spectrometer SUMY in
the Institute of Geological Sciences, the Academy of Sciences
of Belarus in Minsk. The isotope ratios were measured in car-
bon dioxide obtained by way of reaction of the analysed sam-
ples with 100 % orthophosphoric acid. The carbon dioxide was
subsequently trapped in liquid nitrogen and purified in a vacu-
um. The analytical error for single measurements is ±0.2 .
Stable carbon isotope ratios in the carbonate samples are pre-
sented here vs. PDB standard while stable oxygen isotope ra-
tios vs. both PDB and SMOW standards (the latter values are
given in brackets) to simplify the conducted calculations.
Some portions of each analysed calcite sample did not come
directly from the surface, but from the subsurface part of the
pisoids. It is due to the fact that, for technical reasons, the min-
imal mass of a calcite sample required for defining the content
of
18
O is 10 mg. This resulted in contamination of the analysed
portion with the portion of calcite which had been precipitated
earlier (cf. Fig. 3). However, on the basis of insignificant year-
to-year fluctuation in temperature (Wigley & Brown 1976)
within caves as well as stable isotope composition in cave wa-
ter (see Harmon 1979; Yonge et al. 1985), one can assume that
it should not influence the measured stable isotope composi-
tion of the calcite.
Fig. 4. Porous mucilaginous biofilm covering the surface of cave
pisoid.
Fig. 3. Cross-section of pisoid from Perlová Cave, location of sample
taken for measurements of stable isotope composition is indicated.
Fig. 2. Stepped rimstone pools with pisoids, Pearls Passage, Perlová
Cave.
BACTERIAL INFLUENCE ON SPELEOTHEM OXYGEN ISOTOPE COMPOSITION 201
Results
The proportion of stable isotopes in water (D,
18
O
w
) analy-
sed in two series are quite uniform (Table 1). This is in accor-
dance with the observations by Harmon (1979) and Yonge et
al. (1985), who ascertained that the isotopic composition of
percolating water is equal to the average annual isotopic com-
position of the rainwater in the catchment area. Admittedly,
the
δ
D and
δ
18
O
w
values become more negative in the second
series of samples in comparison with the first one. This is no-
ticeable in pools P1 and P5. The shift is probably linked to so-
called seasonal effects (cf. Rozanski & Dulinski 1988). The
majority of the
δ
18
O
c
values obtained from the analysed
pisoids range from 8.3 (22.3 ) to 5.0 (25.8 ),
with the exception of the
δ
18
O
c
value from one pisoid (sample
P6) which is 0.1 (31.0 ; Table 2).
calcite permitted a test of whether the process of crystalliza-
tion proceeded under equilibrium conditions. The test was car-
ried out using the dependence expressed by the equation for-
mulated by ONeil et al. (1969) and modified later by
Friedman & ONeil (1977):
10
3
ln
α
c-w
= 2.78 (10
6
T
2
) 2.89
(1)
where T is the temperature of the process in Kelvin, and the
parameter
α
c-w
is the calcitewater fractionation factor, which
is expressed by equation as follows:
α
c-w
=
&
&
+
+
δ
δ
O
O
c
w
(2)
The values of expression 10
3
ln
α
c-w
for pisoids from Perlová
Cave were calculated using the equation 2 and the measured
values
δ
18
O
w
and
δ
18
O
c
. The results of the calculations
(10
3
ln
α
c-w
) are presented in Table 2. As the values
δ
18
O
w
measured in two series differed slightly from each other the
parallel calculations were made for both of them (in the case
of the pisoids from pools P1, P5, P8 and P9). Since the tem-
perature of crystallization is known, one can compare the ex-
pressions 10
3
ln
α
c-w
and 2.78 (10
6
T
2
) 2.89, which should
be equal according to equation 1. However, the comparison
reveals that these expressions are not equal. The above depen-
dencies are presented in Fig. 5, where it is shown that the cal-
culated values 10
3
ln
α
c-w
fall above the equilibrium line. This
proves that the crystallizing calcite is enriched in the heavy
oxygen isotope in relation to ambient water and that the ma-
jority of the analysed pisoids grew under conditions of dise-
quilibrium between the water and calcite.
Stable oxygen isotope fractionation
during the growth of pisoids in Perlová Cave
Several possible reasons for the disequilibrium conditions
between water and calcite ought to be considered. The dise-
quilibrium can be caused by: (i) rapid diffusion of CO
2
from
solution to atmosphere; (ii) water evaporation; (iii) kinetic ef-
fects connected with the rate of calcite crystallization; (iv) in-
corporation of other (besides Ca
2+
) cations into the calcite
structure, and (v) the effects caused by metabolism of organ-
isms.
The rapid diffusion of CO
2
from water to cave atmosphere
preferentially removes isotopically lighter molecules (C
16
O
2
)
from water (Usdowski et al. 1991). Thus, the above process
should lead to the relative increase of C
18
O
2
content in water.
However, oxygen isotope equilibration between dissolved
CO
2
and H
2
O occurs simultaneously (Usdowski & Hoefs
1990), and can be expressed as follows:
C
18
O
2
+ H
2
16
O ® C
16
O
2
+ H
2
18
O (3)
The systematic enrichment of dissolved CO
2
remaining in
water in
16
O is the effect of the equilibration process operating
during rapid diffusion of CO
2
.
Thus, the above processes should result in the crystalliza-
tion of calcite enriched in
16
O (Guo et al. 1996). The only ex-
Pool
number
Sampling
date
@D
[ SMOW]
@
18
O
[ SMOW]
T
[°C]
T
[K]
1
11.11.1998
69.9
10.51
5.1
278.1
1
19.05.1999
74.5
10.87
5.8
278.8
5
11.11.1998
72.0
10.54
5.1
278.1
5
19.05.1999
74.1
10.85
5.9
278.9
6
11.11.1998
70.9
10.49
4.9
277.9
8
11.11.1998
71.2
10.53
5.0
278.0
8
19.05.1999
70.7
10.43
5.6
278.6
9
11.11.1998
70.2
10.41
4.9
277.9
9
19.05.1999
70.3
10.25
5.8
278.8
10
11.11.1998
72.8
10.70
5.1
278.1
Table 2: Stable isotope composition of water and calcite from stud-
ied pisoids and calculated values of fractionation factor (expressed
as 10
3
lna
c-w
).
Table 1: D and
18
O content in water from Perlová Cave, with mea-
sured temperature values.
Sample
number
@
13
C
[ PDB]
@
18
Oc
[ SMOW]
@
18
Oc
[ PDB]
@
18
Ow
[ SMOW]
10
3
ln=c-w
P 1/1
5.3
25.2
5.5
10.51
35.45
P 1/1
5.3
25.2
5.5
10.87
35.81
P 1/3
3.9
23.8
6.9
10.51
34.09
P 1/3
3.9
23.8
6.9
10.87
34.45
P 1/8
6.3
22.3
8.3
10.51
32.62
P 1/8
6.3
22.3
8.3
10.87
32.98
P 1/9
6.7
23.4
7.3
10.51
33.70
P 1/9
6.7
23.4
7.3
10.87
34.06
P 5/6
7.5
22.6
8.1
10.54
32.94
P 5/6
7.5
22.6
8.1
10.85
33.26
P 6/3
5.0
24.0
6.7
10.49
34.26
P 6/5
4.6
31.0
0.1
10.49
41.07
P 8/2
5.6
25.8
5.0
10.53
36.06
P 8/2
5.6
25.8
5.0
10.43
35.96
P 9/1
6.4
22.7
8.0
10.41
32.91
P 9/1
6.4
22.7
8.0
10.25
32.76
P 9/2
6.5
23.6
7.1
10.41
33.79
P 9/2
6.5
23.6
7.1
10.25
33.63
P 9/4
5.9
23.2
7.5
10.41
33.40
P 9/4
5.9
23.2
7.5
10.25
33.23
P 10/2
6.3
23.1
7.6
10.70
33.59
Assuming that the calcite crystallizes in isotope equilibrium,
the content of
18
O in the calcite depends on the content of
18
O
in the ambient water and on the temperature of crystallization.
The knowledge of the temperature in the cave as well as the
content of
18
O in the ambient water and the content of
18
O in
202 GRADZIÑSKI
ception occurs when chemical reactions leading to the precipi-
tation of the CaCO
3
proceed faster than the process of oxygen
isotope equilibration between CO
2
and H
2
O. Such a process
occurs near springs fed by water overcharged with CO
2
(cf.
Duliñski et al. 1995; Guo et al. 1996), but not in a typical
karst cave, such as Perlová Cave.
Evaporation should be regarded as the second process influ-
encing the content of
18
O in calcite. Evaporation preferentially
removes lighter molecules H
2
16
O from water (Epstein & May-
eda 1953). This leads to the isotope disequilibrium between
water and dissolved CO
2
and HCO
3
. Due to isotope re-equili-
bration the process expressed by equation (3) proceeds from
right to left. The CaCO
3
crystallization under such conditions
results in systematic enrichment in the heavy oxygen isotope
of successively precipitated portions of CaCO
3
. However, the
precipitated CaCO
3
still reflects the changes of
18
O/
16
O ratio
in the water. Because CaCO
3
precipitates in isotopic equilibri-
um with the ambient water, the only exception is in conditions
where crystallization proceeds faster than isotopic re-equili-
bration between dissolved CO
2
, HCO
3
and H
2
O. In such cases
the precipitated calcite would be more enriched in the light
isotope of oxygen than a carbonate system of the ambient wa-
ter (see e.g., Usdowski et al. 1979; Dandurand et al. 1982;
Turi 1986; Chafetz et al. 1991; Chafetz & Lawrence 1994).
Therefore, the above mechanism cannot be applicable to Per-
lová Cave. It is noteworthy, that the relative humidity in deep-
er parts of single-entrance caves situated, like Perlová Cave,
in the temperate climate zone is very high and reaches over
95 % in summer and even 100 % in other seasons (Wigley &
Brown 1976). Thus evaporation in the studied cave seems not
to proceed at all or proceeds on a negligible scale.
The rate of crystallization may influence the isotopic com-
position of calcium carbonate (Chafetz et al. 1991; Dickson
1991; Chafetz & Lawrence 1994). The more rapid the process
of crystallization is, the more calcite is enriched in the light
isotope of oxygen (cf. Hoefs 1997). This leads in the opposite
direction to that detected in the studied pisoids. Therefore, the
rate of crystallization can be excluded from the factors con-
trolling isotope exchange between the ambient water and cal-
cite. The influence of other cations apart from Ca
2+
should
also be excluded, because of the absence of their significant
admixture within the pisoids (cf. ONeil et al. 1969; Mortimer
& Coleman 1997).
Therefore another explanation is to be sought. The most
plausible explanation deals with the physiology of the bacte-
ria. Bacterial physiology may cause isotopic disequilibrium
between water and calcite. Organisms show a metabolic pref-
erence for the light rather than the heavy isotope (Ehrlich
1996, 1998). The phenomenon is known mainly in relation to
stable carbon isotopes (cf. Mertz 1992 and references quoted
herein). Preferential use of the light oxygen isotope is decid-
edly less known. Such effects have been found in foraminifer-
al tests and coral skeletons (Grossman 1987; McConnaughey
1989). As for the foraminifers it is demonstrated in the enrich-
ment of tests in the light isotope of oxygen (
16
O) due to the in-
corporation of isotopically light metabolic oxygen molecules
into the test (Grossman 1987). Recently, examples of fraction-
ation of the oxygen isotopes caused by the metabolic process-
es of bacteria have also been reported. Mortimer & Coleman
(1997) showed the influence of the bacteria Geobacter metal-
lireducens on the
δ
18
O values of diagenetic siderite. Horita et
al. (1998) also reported a similar process in the iron oxides of
microbial origin.
As mentioned above, hydrogen-oxidizing bacteria occur in
the biofilm covering the studied pisoids. The bacteria change
the chemistry of their microenvironments by uptake of oxy-
gen either in the form of O
2
, which they use to obtain energy
from reaction of the synthesis of the water, or in the form of
CO
2
used to build up the organic matter (Aragno & Schlegel
1992).
Oxygen isotope disequilibrium can be caused by the prefer-
ential uptake of the C
16
O
2
molecules and/or by the preferen-
tial uptake of
16
O
2
. It is not possible to determine which pro-
cess is of crucial significance. Each can result in the relative
depletion of
16
O
2
in the bacterial microenvironment, that is
within the biofilm. Since the biofilm is a zone isolated from
the external environment (Decho 2000) this depletion is not
sufficiently quickly re-equilibrated. Therefore, the calcite
which grow around the bacterial cells within the biofilm is de-
pleted in the light isotope of oxygen.
The
δ
13
C values of the studied pisoids fall in the range 6.7
to 3.9 (Table 2). It means that they do not differ from values
typical of carbonate speleothems (cf. Schwarcz 1986; Baker et
al. 1997). Unfortunately, it was impossible to carry out the
study of carbon isotopic composition of carbonate molecules
in the ambient solution. It was due to technical reasons (the
relatively small volume of the studied pools and relatively big
amount of water needed for analysis). Therefore, one cannot
decipher the fractionation of the stable isotopes of carbon be-
tween the carbonate molecules in the ambient water and the
growing pisoids.
The differences in
δ
18
O
c
values between the pisoids grow-
ing in the single pool are another question which can be
posed. They probably owe their origin to the variable intensity
of bacterial physiological processes existing on a microenvi-
ronmental scale and their intensity can change over a very
short distance (cf. Chafetz et al. 1991). Local mechanical deg-
radation of a biofilm, which can cause faster re-equilibration
of the stable isotope composition between the biofilm and ex-
ternal environment, seems to be another explanation.
Fig. 5. Fractionation factor (expressed as 10
3
ln
α
c-w
) vs. temperature
(expressed as 10
6
T
2
); triangles values calculated for temperatures
of November, squares values calculated for temperatures of May.
BACTERIAL INFLUENCE ON SPELEOTHEM OXYGEN ISOTOPE COMPOSITION 203
Conclusions
This study suggests that the physiology of chemo-
lithoautotrophic hydrogen-oxidizing bacteria accounts for ex-
change of stable oxygen isotope occurring during the growth
of pisoids in Perlová Cave. This indicates that not only CO
2
diffusion and evaporation but also biological processes influ-
ence the oxygen isotope ratio within speleothems. It can be
assumed that these processes also have an effect on other spe-
leothems attributed to a microbial origin (Le Métayer-Levrel
et al. 1997; Northup & Lavoie 2001; Jones 2001). This sug-
gestion especially concerns moonmilk speleothems, which are
also formed by mediation of hydrogen-oxidizing bacteria
(Gradziñski et al. 1997).
Acknowledgments: The study is a part of the authors PhD
thesis prepared under the supervision of Prof. A. Radomski
(Institute of Geological Sciences, Jagiellonian University,
Kraków, Poland). The author wishes to thank Peter Holúbek
for field assistance, Jadwiga Faber for taking SEM photo-
graphs, and to Dr. Marek Duliñski (Faculty of Physics and
Nuclear Technics, University of Mining and Metallurgy,
Kraków, Poland) for discussion. Thanks also go to Dr.
Joachim Szulc (Institute of Geological Sciences, Jagiellonian
University) for constructive comments on an early draft of
this paper. The study was financed by the State Committee for
Scientific Research (KBN) Grant No. 6PO4D 019 14. M.G. is
supported by the Foundation for Polish Sciences (Prof. J.
Kamierczak Grant for Researchers). Criticism of Dr. Pavel
Bosák (Institute of Geology, Czech Academy of Sciencs), Dr.
Micha³ Gruszczyñski (Institute of Paleobiology, Polish Acad-
emy of Sciences, Warszawa, Poland) and two anonymous re-
viewers helped to improve the manuscript.
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