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Geochimica et Cosmochimica Acta 75 (2011) 4441–4455

www.elsevier.com/locate/gca

Biotic and inorganic control on travertine deposition at

Bullicame 3 spring (Viterbo, Italy): A multidisciplinary approach
F. Di Benedetto a,b, G. Montegrossi c,⇑, A. Minissale c, L.A. Pardi d, M. Romanelli a,
F. Tassi b, A. Delgado Huertas e, E.M. Pampin f, O. Vaselli b,c, D. Borrini b
a
Dip. Chimica, Università di Firenze, Via della Lastruccia, 3, 50019 Sesto F.no, Italy
b
Dip. Scienze della Terra, Università di Firenze, Via G. La Pira, 4, 50121 Firenze, Italy
c
CNR-IGG Istituto di Geoscienze e Georisorse, Via G. La Pira, 4, 50121 Firenze, Italy
d
CNR-IPCF Istituto per i Processi Chimico-Fisici, Via Moruzzi, 1, 56010 Pisa, Italy
e
Estación Experimental del Zaidı́n (CSIC), Prof. Albareda, 118008 Granada, Spain
f
Genetica (Produccion Animal), Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

Received 3 June 2010; accepted in revised form 6 May 2011; available online 18 May 2011

Abstract

A multidisciplinary characterization of an active thermal spring in central Italy has been undertaken with the aim of (i)
ascertaining whether microbiological activity plays a relevant role in hot-depositing travertines and (ii) establishing an exper-
imental protocol able to identify similar effects in fossil travertines. Water, gas, and travertine samples were investigated by
chemical (ICP/MS, SEM/EDS), physical (DTA–DTG), isotopic (d18O, dD, and d13C), mineralogical (XRPD), and spectro-
scopic (EPR) techniques. Twenty-four samples (three for each phase) were collected every 5 °C temperature drop, along a
100 m long artificial channel near Viterbo (Bullicame 3, Latium, central Italy). A microbiological characterization was car-
ried out in parallel, sampling the channel every 10 °C temperature drop.
The Bullicame 3 system is revealed to be composed of two markedly different subsystems: a water/gas interface, where a
kinetically fast exchange allows equilibrium of components both in water and in gases; a solid/water interface, where traver-
tine precipitation occurs, influenced by microbiological activity. A peculiar lattice shrinking of calcite was identified, as well as
an anomalous value of the zero-field splitting parameter from the EPR measurements. The interpretation of these anomalies is
confirmed by the identification of calcifying cyanobacteria throughout the channel path.
Our results point out that microbiological activity can play a significant role in travertine deposition from hot springs. Fur-
thermore, the proposed approach, representing a tool to identify crystal chemical remnants of past microbiological activity,
could be applicable to fossil travertines.
Ó 2011 Elsevier Ltd. All rights reserved.

1. INTRODUCTION (e.g. Andrews, 2006), neotectonics and paleohydrology (e.g.

In the last decades thermogene and/or meteogene (tufa)
travertine deposits have received an increasing interest, par-
ticularly after the discovery of the involvement of bacteria
in their formation (Chafetz and Folk, 1984) and the role
they played in the appearance of life on the planet (Farmer,
2000). Travertine deposits are important geological materi-
als since they are used as: (i) proxy in climate change studies
⇑ Corresponding author. Tel.: +39 055 275 7477.
E-mail address: montegrossi@igg.cnr.it (G. Montegrossi).
Hancock et al., 1999; Minissale et al., 2005); (ii) geochemi-
cal repository of pollutants (e.g. Di Benedetto et al., 2006);
and (iii) building stones involving technological and cul-
tural heritage (Attanasio, 1999).
The role of biological activity (e.g. bacteria, algae, micro-
organisms, and so forth) in travertine formations is the focus
of a close scientific debate, namely concerning whether the
presence of biotic activity could hinder the reconstruction
of paleoclimatic environments (Andrews and Riding, 2001;
Fouke, 2001). Two opposite approaches have been pro-
posed: (a) Guo et al. (1996), by studying the Rapolano (Italy)

0016-7037/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.gca.2011.05.011
4442 F. Di Benedetto et al. / Geochimica et Cosmochimica Acta 75 (2011) 4441–4455
active springs (low temperature, T = 29–34 °C), suggested
microbial activity to significantly affect the carbon isotopic
signature (13C/12C ratio increases downstream), probably
due to the preferential metabolization of 12CO2 by living
organisms; (b) Fouke et al. (2000), in a study of the Angel
Terrace springs (Yellowstone Park, USA; T = 71–73 °C),
suggested a totally inorganic process controlling the fraction-
ation of 12CO2 during travertine deposition.
In recent studies, two main topics were considered. On
the one hand, theoretical studies showed that a non-
equilibrium isotopic signature cannot fully discriminate be-
tween concurring kinetically-driven and biogenic deposi-
tions (Skidmore et al., 2004; Scholz et al., 2009). On the
other hand, Pokroy et al. (2006) and Zolotoyabko et al.
(2010) identified structural and chemical evidences in bio-
genic calcite, namely: (a) the presence of a permanent lattice
strain (elongation along the c-axis) unrelated to the chemi-
cal composition of calcite (in particular to the occurrence of
cation/anion replacement) and (b) the presence of biogenic
sulfur, i.e. S-bearing biomolecules co-precipitated or
embedded in the carbonate fabrics.
Moreover, Lee et al. (2006) discussed the capability of the
Sinechococcus cyanobacteria of bio-precipitating calcite, and
the effects of external chemical and physical constraints (e.g.
carbonate/bicarbonate availability, pH) on the bacterial
activity. Montegrossi et al. (2006), in a study on some Italian
travertine deposits, revealed an unusual variability of the
zero-field splitting parameter by Electron Paramagnetic Res-
onance (EPR) spectroscopy, suggesting that chemical order-
ing of cations in travertine can be investigated, as already
proposed in the pioneering study by Kretz (1982). Calcite is
known, in fact, to retain many non-equilibrium depositional
features (e.g. Wang and Merino, 1992; Klein and Hurlbut,
1993; Humphrey and Howell, 1999).
A multidisciplinary study was undertaken with the aim of
exhaustively monitoring the gas/water and water/solid inter-
faces in an active thermal spring (named Bullicame 3; 57 °C,
according to Duchi et al., 1985; otherwise cited as “Piscina
Carletti”), located north of Rome, close to the Etruscan city
of Viterbo (central Italy), in order to gain complete informa-
tion on the physical, chemical and biological parameters.
Bullicame 3 represents an excellent natural laboratory to
investigate the parameters acting on the travertine precipita-
tion process, such as (i) temperature, (ii) CO2, and (iii) bacte-
rial activity (Pentecost, 2005). Physical, chemical, and
isotopic features of the gas, liquid, and solid phases were
determined, and the resulting information was combined
with lattice deformation, thermal analysis (DTA–DTG)
and Mn(II) EPR measurements of the travertine samples.
Moreover a thorough investigation of the microbiological
species present in the thermal water was performed.
2. SAMPLING AREA AND EXPERIMENTAL
PROCEDURES
2.1. Location and brief outlines of the Bullicame thermal
discharge system
Bullicame (42.25N/12.03E) is a thermal area located
3 km west of the Etruscan and after papal city of Viterbo
(northern Latium, Italy), hosting several hot water
discharges in a small homonymous natural park.1 One of
them, known as Bullicame 3 (Duchi et al., 1985; Pentecost,
1995; Minissale et al., 2002), is artificially elevated over the
ground few meters and it is canalized to form a simple
open-air Roman-style thermal bath. The thermal discharge
(57 °C) is confined in a 3 m in diameter pool and is accom-
panied by a vigorously bubbling gas (CO2-rich) phase. A
relatively narrow (14 cm) channel allows the thermal water
to gently flow for about 112 m to the end of the channel,
where a cooler pool (frigidarium) is equilibrated with the
ambient temperature (Fig. 1). All artificial structures, chan-
nels and pools, made up by bricks, are covered by a 10–
20 cm thick travertine deposit.
The Bullicame thermal spring discharges have been
known since the Etruscan age. The Etruscan and the Ro-
man civilizations were particularly attracted by thermal
baths and, consequently, fostered the development of this
area. The first documented evidence of the use of this loca-
tion as a spa resort dates back to the I–II century B.C.,
although it was not associated to any important conurba-
tion, but likely related to private country houses; urban set-
tlements are indeed described in the VII century B.C.
(Giannini, 1969). Successively, Viterbo and the Bullicame
site were developed by Papal State during the XIII century
AD. This thermal area is also cited by Dante in the Divina
Commedia: “Quale del Bullicame esce ruscello/che parton poi
tra le lor peccatrici/tal per la rena giù sen giua quello./Lo
sfondo suo ed ambo le pendici/fatt’eran pietra, è margin di
lato” (Dante Alighieri, Hell XIV, 79–84).
The spring of Bullicame 3, already studied in detail by
Duchi et al. (1985) and Pentecost (1995), has a flow section
of 150 cm2 with a relatively constant water speed (7.7 cm/s)
and flow rate (0.7 L/s). Travertine fabrics are constant
throughout the whole channel and can be described in
terms of the “shrub” assemblages, already pointed out by
Pentecost (1995) and Guo et al. (1996) (Fig. 2).
Eight samples (Tr1–Tr8) of travertine, water, and gas
were collected along the channel, in which temperature var-
ies between 57 and 22 °C. The sampling was performed at
constant temperature decrease of 5 °C (Fig. 1b). An addi-
tional gas sample (Tr0) was taken at the thermal spring dis-
charge (i.e. the free bubbling gas at the center of the spring,
Fig. 1b).
2.2. Gas sampling and analysis
The free gas phase at the spring was collected by using
an upside-down funnel connected to a pre-evacuated
60 mL glass flask equipped with a Teflon stopcock and
filled with 20 mL of a 4 N NaOH and 0.15 M Cd(OH)2 sus-
pension (Montegrossi et al., 2001).
Water samples for the analysis of the dissolved gases
were collected into one-way, pre-evacuated 125 mL glass
flasks equipped with Teflon valves that were filled with
1
As reported in the may 2010 volume (Italian edition) of
National Geographic, the thermal spring and the park are presently
threatened by the project of a large airport that should be realized
in the area.
 Biotic and inorganic control on travertine deposition
Fig. 1. (a) Aerial photograph and map of the investigated area and (b) location of the travertine, water, and gas samples.
Fig. 2. SEM micrograph of a travertine fragment.
50–60 mL of channel water (Tassi et al., 2008). The deter-
mination of the gas species in the head-space was per-
formed by gas-chromatography (Vaselli et al., 2006), and
the total amount of the dissolved gases was calculated
according to Henry’s law. The contents of CO2, N2, O2,
H2S, CH4, Ar, Ne, and H2 were determined using Shimadzu
14A and 15A gas-chromatographers equipped with Flame
Ionization Detector (FID) and Thermal Conductivity
Detector (TCD), respectively. The 13C/12C isotopic ratio
of CO2 (expressed as d13C & V-PDB) was determined by
a Finnigan Delta S mass spectrometer after a two-step
extraction and purification procedure of the gas mixtures
4443
by using liquid N2 and a mixture of liquid N2 and trichlo-
roethylene (Evans et al., 1988; Vaselli et al., 2006). Internal
(Carrara and San Vincenzo marbles) and international
(NBS18 and NBS19) standards were used for the estimation
of external precision. Analytical error for d13C values is
±0.05&.
2.3. Water sampling and analysis
Two aliquots of filtered (at 0.45 lm) water samples were
collected in 125 and 50 mL plastic bottles (after adding
0.5 mL of concentrated HNO3), for anion and cation anal-
ysis, respectively. Unfiltered water (20 mL) was collected in
glass bottles for isotopic analysis (O, H, and C-DIC, Dis-
solved Inorganic Carbon) after adding few mg of HgCl2
to prevent any microbial activity. Temperature, pH,
HCO3 (acidimetric titration) and NH4+ (Nessler colori-
metric method) contents were determined in situ. The water
samples were analyzed with a Dionex DX100 ion chro-
matograph (F, Cl, Br, NO3, and SO4) and a Perkin-Elmer
AAnalyst 100 atomic absorption spectrophotometer (Na,
K, Ca, Mg, Fe, and Mn); dissolved CO2 concentrations
were determined via acidimetric titration (Tassi et al.,
2004).
Oxygen isotopes were analyzed by using the CO2–H2O
equilibration method (Epstein and Mayeda, 1953). The
hydrogen isotopic measurements were carried out by
reducing the water sample to hydrogen with hot zinc at
550 °C, according to the analytical procedure described
by Coleman et al. (1982). Oxygen and hydrogen isotopic
4444 F. Di Benedetto et al. / Geochimica et Cosmochimica Acta 75 (2011) 4441–4455
compositions (expressed as d18O and dD & V-SMOW,
respectively) were determined using a Delta Plus XL and
MAT 251 (Finnigan) mass spectrometers. The analytical er-
ror for d18O and dD values was ±0.1& and ±2&, respec-
tively. The d13C values of Dissolved Inorganic Carbon
(DIC) were determined with the same mass spectrometer
used for the d13C–CO2 analysis, after the reaction of
3 mL of water with 2 mL of anhydrous phosphoric acid
in a under-vacuum line (Salata et al., 2000). The recovered
CO2 was analyzed after the two-step extraction and purifi-
cation procedures. Analytical error for d13C was ±0.05.
2.4. Travertine samples
Travertine fragments were sampled from the bottom of
the channel at the contact point with the collected waters
and dissolved gases. Samples were obtained after removing
the encrustations, and then gently powdered in an agate
mortar after drying at 40 °C.
Chemical analyses of travertine samples were performed
at the Acme Analytical Laboratories Ltd. (Vancouver,
Canada), after aqua regia digestion, by means of Atomic
Absorption Spectrophotometry (AAS) and Induced Cou-
pled Plasma–Mass Spectrometry (ICP/MS), whereas C
and S were analyzed by Leco Gas chromatography with
an IR detector.
X-ray powder diffraction (XRPD) patterns were ob-
tained using a Philips PW 3710 diffractometer with Cu anode
and graphite monochromator, equipped with PC-X’Pert Pro
software for data acquisition and handling. Experimental
conditions were 20 mA current, 40 kV voltage, 20–80° 2h
range, step size 0.02° 2h, 2.5 s per step. The powder struc-
tural refinement was carried out through the Rietveld algo-
rithm using the Rietquan software (Lutterotti et al., 1998).
Thermal factors for Ca, C, and O are from Markgraf and
Reeder (1985). A reference synthetic calcite sample (Rudi
Pont, Turin, Italy) was used as external standard for the dif-
fractometer calibration (lattice constants, peak shape, and
2h offset). A raw travertine encrustation was also analyzed
without pre-treatment, in order to check whether the grind-
ing procedure would favor the conversion of aragonite onto
calcite (e.g. Criado and Trillo, 1975).
Conventional EPR spectroscopy measurements were
performed on free powdered samples, in amorphous silica
tubes. Data were collected at room temperature using a
Bruker ER 200D-SRC spectrometer operating at X-Band
(9.5 GHz) interfaced with DS/EPR software to a PC for
data acquisition and handling. The actual operating
frequency value was determined by using DPPH radical
[2,2-di(4-tert-octyl-phenyl)-1-picrylhydrazyl, g = 2.0037] as
external standard. Due to the narrow line-width of Mn(II)
spectra in calcite (Vassilikou-Dova, 1993), particular care
was paid to the instrumental field modulation and sweep
rate. The chosen parameters were 0.5 G modulation ampli-
tude and 100 kHz modulation frequency. Scan speed was
set to 2 G/s. The full Mn(II) spectrum was preliminarily
identified in each sample, in the magnetic field range
2800–4000 G. Successively, a detailed spectrum was
recorded in the range 3600–3700 G, where the sixth hyper-
fine line of the central ½ ) ½ transition appears. The
Fig. 3. EPR derivative plot (arbitrary units) of the sixth hyperfine
line of the Mn(II) spectrum, putting in evidence the relevant points
for the D parameter evaluation. In the inset, second derivative plot
of the same spectrum, putting in evidence the relevant points for
the WD parameter evaluation. Magnetic field values are expressed
in Gauss.
difference between the field positions corresponding to the
relative maximum and minimum of this partial spectrum
gives the D6 parameter (Montegrossi et al., 2006).
Subsequently, after a smoothing of the raw spectrum to re-
duce the experimental noise, a numerical derivative curve
was calculated. The difference between the field positions
corresponding to the two relative maxima (Fig. 3) yields
the W6 parameter. The D6 and W6 parameters can be re-
lated, via the quantification of the zero-field interaction,
to the strength of the crystal field and to the structural dis-
order, respectively. These values were converted to the cor-
responding D (most frequent value of the axial ZFS
parameter) and WD (distribution of the axial ZFS interac-
tion) values by means of the following relations2:
pffiffiffiffiffiffi
D ¼ 0:24294 D6 ð1Þ
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
W D ¼ 0:48588  W 6  D6 ð2Þ
Scanning electron micrographs (up to 104 magnifica-
tion) of selected samples were obtained by using a SEM
Philips 515 microscope interfaced to a personal computer
through a digital acquisition software. Point and raster mi-
cro-analytical data were obtained by means of an energy-
dispersive analyzer and an EDAX 9800 analytical system.
A constant beam was maintained during the investigations,
the current and voltage being 20 nA (on the sample) and
25 kV, respectively.
Thermal analyses were performed on a Netzsch STA 409
apparatus (accuracy ±3 °C) on finely ground travertine
samples. Pure fine grained (<63 lm) alumina, a-Al2O3,
was used as reference standard. Powders were contained
in alumina open crucibles, heating rate was 3 °C/min, max-
imum programmed temperature was 280 °C, focusing on
2
Relations (1) and (2) are defined for D, WD, D6, W6 values
expressed in Tesla (1T = 10000 G), according to Montegrossi et al.
(2006).
 Biotic and inorganic control on travertine deposition 4445

the temperature range of sulfur melting. Besides an Al2O3/

CO2(aq)
Al2O3 run, to account for instrumental effects, temperature

9.68
6.91
6.94
4.85
4.63
2.99
3.22
2.96

6.80
and calorimetric calibration were also performed, using

–
low-melting pure standard substances, namely phenan-

 a
threne (C14H10; 99.3 °C), elemental In (156.61 °C) and ele-

166
153
151
138
142
132
130
129
190
F

–
mental Se (217 °C).

 a
2.5. Isolation of microorganisms

113
107
110
83
71
77
81
80
HS

–
–
The presence of fungi, bacteria and phytoplankton was

NO3 a
investigated in four water samples having different temper-

100.8
12.5
12.9
28.2
84.7
2.4
2.4
1.8

All values are expressed in meq/L, with the exception of those analytes marked with superscript ‘a’ and CO2(aq) that are in leq/L and mmol/L, respectively.
atures (57, 47, 37, and 27 °C, respectively). One hundred

–
–
microliters of each water sample was placed in Petri dishes

 a
with two culture media: Sabouraud Dextrose Agar (Bio-

0.94
0.78
0.91
0.94
1.10
1.10
0.94
1.10
Br
merieux, Madrid, Spain) and Columbia Blood Agar (Bio-

–
–
merieux, Madrid, Spain) for the isolation of fungi and

2
bacteria, respectively. The incubation was performed at

21.88
21.88
21.88
22.92
22.92
23.96
21.88
20.83
23.00
22.8
SO4
25 and 37 °C. Yeast identification was made by macro-

Physical–chemical features of the Bullicame 3 thermal spring discharge and of the water samples collected along the artificial channel.
scopic observation and microscopic morphology, by apply-

Chemistry (anions)

0.416
0.416
0.409
0.367
0.430
0.451
0.367
0.388
0.400
ing standard taxonomic criteria (de Hoog et al., 2000).

0.40

Cl
Bacteria identification was made by macroscopic and
microscopic observation, and by simple biochemical tests,


applying standard taxonomic criteria (Quinn et al., 2000).

HCO3
16.08
16.17
16.11
13.69
12.66
11.64
11.96
11.01
15.60
14.6
Microalgae were identified in fixed samples (4% PBS-buf-
fered formalin) in settling chambers using an inverted
microscope (Axiovert 35, Zeiss, Oberkochen, Germany).
+ a

18.7
18.7
20.2
18.7
17.3
18.7
18.7
18.7
21.3
The identification of algae was carried out in accordance
Li

–
with John et al. (2002) procedure. NH4+ a

860.0
25.7
17.9
15.0
7.1
3.6
3.6
3.6
3.6
2.6. Theoretical modeling of isotopic signatures

–
Measured isotopic signatures were modeled through the-
0.86
0.90
0.88
0.94
0.91
0.91
0.89
0.89
0.88
0.85
+

oretical calculations, performed on the basis of (i) measured

K

d13C-DIC ratios, (ii) temperature, and (iii) bulk chemical

composition of waters, gas, and travertine. Numerical simu-
+

1.39
1.37
1.34
1.35
1.39
1.43
1.39
1.41
1.52
2.80
Na

lations of transport and isotope fractionation provide, in

Chemistry (cations)

fact, a method to quantitatively interpret isotope fraction-

2+

9.38
9.71
9.88
9.71
9.96
9.88
9.63
9.88
9.20
ation profiles based on evaporation, gas diffusion and calcite
11.5
Mg

precipitation. The temperature-dependent equilibrium con-

stants of the considered isotopic species, occurring in the dif-
23.55
23.50
23.60
23.15
22.55
24.05
20.30
20.45
26.40
2+

ferent phases of the system, (taken from the database of

28.0
Ca

PHREEQC, Parkhurst and Appelo, 1999) were included

into the thermodynamic database of the reactive transport
6.54
pH
6.2
6.7
7.3
7.6
7.8
7.9
7.7
7.8
6.6

code TOUGHREACT (Xu and Pruess, 2001; Spycher and

Pruess, 2005; Xu et al., 2006), by means of the EOS2 model.
The different diffusive transport properties of the considered
T (°C)

56.3

species (Palmer, 1994; Tamini et al., 1994; Frank et al., 1996)

57
52
47
42
37
32
27
22
57

and water density dependence on NaCl and CO2 (Garcia,

Physical features

2001) were also considered. These calculations have been

Distance (m)

used to illustrate the evolution of stable isotope profiles

along the channel, i.e. outside the spring pool (Fig. 1b).
26.34
50.14
56.54
60.84
67.44
80.04
94.64

The channel was modeled by an array of 99  30  31

0

0
0

elements, each one having dimension 1.0  0.005 

0.005 m3, respectively. The bottom and lateral boundaries
Duchi et al. (1978)

(walls of the channel) are simulated by two layers of calcite

Pentecost (1995)

elements (porosity: 6%, permeability: 1015 m2). The upper

boundary layer simulates atmosphere, having a fixed buffer
of 400 lmol/mol of CO2. This layer also plays the role of
Table 1

Sample

thermal buffer, being maintained at a constant temperature

Tr1
Tr2
Tr3
Tr4
Tr5
Tr6
Tr7
Tr8

of 15 °C. An in-flow is located at the warmer side of the

4446 F. Di Benedetto et al. / Geochimica et Cosmochimica Acta 75 (2011) 4441–4455

Table 2
Dissolved gas samples composition (in % by vol.) from the thermal spring discharge to the end of the artificial channel at Bullicame 3.
Sample O2 N2 CO2 Ar Ne
Tr0 (emission) 0.17 1.12 98.66 0.02 0.000012
Dissolved gas samples
Tr1 6.71 12.76 80.21 0.321 0.00019
Tr2 15.19 29.92 54.15 0.746 0.00050
Tr3 15.73 30.93 52.56 0.773 0.00052
Tr4 16.28 31.95 50.98 0.800 0.00053
Tr5 17.77 35.48 45.86 0.892 0.00053
Tr6 17.75 35.48 45.89 0.878 0.00059
Tr7 18.11 36.18 44.82 0.892 0.00060
Tr8 25.99 51.97 20.76 1.278 0.00078

simulation array, according to the experimental values for

flux and velocity. At the cooler side of the array, the
out-flow is simulated as an extraction well. The initial con-
ditions (at the warmer side) are set up according to the
chemical and physical data obtained for sample 1 (Table 1).
The system is then allowed to evolve, simulating the flow
through the channel.

3. RESULTS

3.1. Water and gas chemistry

The Bullicame spring system is characterized by a Ca-

SO4-HCO3 composition typical of most waters circulating
in the Mesozoic Carbonate Sequence belonging to the Tus-
can Nappe (e.g. Minissale, 2004). The temperature and the
chemical composition of the Bullicame 3 spring, determined
in the present study, is fully comparable to that reported by
Duchi et al. (1985). This compositional stability over such a
long time must be ascribed to the relatively long circulation
path of the thermal fluids. These, in turn, originate mainly
from meteoric precipitations gathered inside the main
Apennines Range (e.g. Minissale, 2004).
The composition of the free bubbling gas phase at the
emergence pool of the spring is dominated by CO2
(98.66 vol.%; Tr0 sample) with minor N2 (1.12% by vol.;
Table 2), a common feature of thermal springs in central
Italy (Minissale, 2004). The dissolved gases in this pool
(sampled few centimeters away from the emergence) are
characterized by significantly lower CO2 (80 vol.%; Tr1
sample) and higher N2 (12.7 vol.%) and O2 (6.71 vol.%)
amounts (Table 2). These compositional trends are likely
caused by the dissolution of atmospheric components
(air) in the water according to the Henry’s law. However,
a dissolved gas contribution related to a hypothetical pho-
tosynthetic activity in the pond cannot be ruled out.
Water samples collected along the channel show a pro-
gressive decrease of the dissolved CO2 concentrations from
9.7 to 3.0 mmol/L (Table 1 and Fig. 4a), from the discharg-
ing area to the end of the channel. A similar decreasing pat-
tern is also observed for [HCO3], whose content varies
from 16.08 down to 11.01 meq/L. The pH increases from Fig. 4. (a) pH values and concentration of the carbon (HCO3 in
6.22 to 7.79. Such chemical–physical changes are likely meq/L and dissolved CO2 in mmol/L) species; (b) d13C (& V-PDB)
due to both CO2 degassing and travertine precipitation, values of Dissolved Inorganic Carbon (DIC) and of dissolved CO2,
the latter being responsible for a decrease of the Ca concen- calculated (continuous line) d13C of dissolved CO2; (c) relative
trations, from 23.55 to 20.45 meq/L. A further significant content of CO2 (in % by vol. in dissolved gas).
 Biotic and inorganic control on travertine deposition 4447

Table 3
Isotopic ratios from water samples (dD, d18O, and d13C of Dissolved Inorganic Carbon), from gas samples (d13C of dissolved CO2), and from
travertine samples (d13C and d18O).
Sample Water Gas (CO2) Travertine
dD & V-SMOW d18O & V-SMOW d13C DIC & V-PDB d13C-CO2 & V-PDB d13C & V-PDB d18O & V-PDB
Tr1 38.56 6.25 1.16 3.35 8.24 18.20
Tr2 38.91 6.16 2.80 3.44 6.36 20.11
Tr3 37.97 5.95 3.63 3.19 6.06 20.05
Tr4 37.79 5.78 4.04 2.19 6.17 20.13
Tr5 36.79 5.43 4.74 2.14 5.95 19.74
Tr6 36.32 5.23 5.09 0.97 6.00 20.11
Tr7 35.69 5.16 4.88 3.60 6.28 21.43
Tr8 35.36 5.03 5.27 0.72 6.36 21.20

more positive with decreasing temperature (Table 3 and

Fig. 5. Measured and calculated d13C (& V-PDB) values versus
temperature (°C) in travertine samples.
compositional variation along the channel is represented by
the progressive decrease of NH4+ and HS contents, cou-
pled with the simultaneous increase of [NO3] and
[SO42] (Table 1). This can be interpreted as a consequence
of the non-stoichiometric oxidation of the reduced species
to the most stable oxidized species. No other significant
variations of water and dissolved gas chemistry are ob-
served (Table 1).
3.2. Isotopic signature of water, gas, and travertine
In water samples, the dD and d18O values become pro-
gressively less negative along the channel (Table 3). In trav-
ertine encrustations d18O values moderately increase with
decreasing temperature (Table 3). This behavior is typical
of travertine-depositing thermal springs with temperature
>35–40 °C (Gonfiantini et al., 1968; Fouke et al., 2000).
The variation of d18O in water (from 6.25& to 5.03&
versus V-SMOW) and travertine (from 18.2& to 21.2&
versus V-SMOW) corresponds to that found for the traver-
tine–water fractionation process (Thorstenson and
Parkhurst, 2002). The d18O values show an inverse correla-
tion with both the total amount of dissolved CO2 (Fig. 4a
and Table 1) and its relative abundance (CO2 vol.%) in
the dissolved gases (Fig. 4c and Table 2). Moreover, the
measured d13C ratios of DIC and dissolved CO2 become
Fig. 4b). Thus, both observations suggest that a fast ex-
change with atmospheric CO2 is occurring.
Measured values of d13C in travertine (Fig. 5) abruptly
decrease from an initial value >8 & at 57 °C down to a
nearly constant value of about 6.2&. The lack of a direct
correlation between d13C of dissolved CO2 and d13C in trav-
ertine is a clear fingerprint of an out-of-equilibrium frac-
tionation process. In order to have an insight into this
process, we performed sample calculations, as described
above. Calculated d13C data of dissolved CO2 (Fig. 4b)
and of travertine (Fig. 5) agree with the experimental ones
along the whole series but for the first point.
The calculation results show that diffusion of carbonate
species in solution from water–atmosphere to water–traver-
tine interface is the rate determining step. The calculation,
in fact, generates vertical concentration profiles of dissolved
CO2, as shown in Fig. 6a for the field samples. Isotopic sig-
natures of three levels in the vertical profiles of the channel
(i.e. the bottom, 5 cm and top levels) are shown in Fig. 6b.
Lighter dissolved CO2 is almost quantitatively preserved at
the bottom of the system, whereas the top layer readily at-
tains a steady isotopic ratio, due to a balance between
degassing of lighter CO2, dissolution of atmospheric gases
and diffusion of CO2 in the water phase. An enhanced effect
of the preferential degassing of lighter CO2 is evidenced by
the ratios calculated at 5 cm (from bottom of the channel),
where the combined effects of the mass transport and isoto-
pic exchange lead to a progressive increase of d13C value
downstream (see e.g. Chafetz et al., 1991). The relative sta-
bility of the bottom layer of the system (Fig. 6a and b) re-
sults in a small variation of isotopic composition of the
depositing calcite (Fig. 5, calculated values). According to
this calculation, therefore, a full inorganic process could
be operative in the system.
3.3. Mineralogical and chemical composition of the travertine
deposits
Travertine encrustations are characterized by the mas-
sive presence of CaCO3 that occurs as two main poly-
morphs: calcite and aragonite, although the former is by
far the most abundant (>94 wt%). Pentecost (1995) points
out the presence of strontianite (SrCO3), quartz and gyp-
sum at Bullicame 3 but in the present study they were
always found to be below the detection limit of 1.0 wt%
4448 F. Di Benedetto et al. / Geochimica et Cosmochimica Acta 75 (2011) 4441–4455
Fig. 6. (a) Calculated vertical profiles of CO2 content (mmol) versus temperature (°C) in the channel; (b) calculated profiles of d13C versus
temperature (°C) for selected depths in the channel.
(Table 5). The low aragonite/calcite ratio (0.06/0.01) in the
present XRPD patterns is likely due to a partial intercon-
version between the two calcium carbonate polymorphs
(Criado and Trillo, 1975). A XRPD characterization of
an uncrushed fraction of travertine sample was thus per-
formed, and no significant variations in terms of relative
amounts of the two minerals were detected.
The chemical composition of all travertine samples is al-
most constant (Table 4) and is dominated by calcium (up to
56 wt% as CaO: Tr5) and CO2 (42.2%: Tr4). The contents
of Mg (as MgO), Sr (as SrO) and S range from 0.8 to 1.0,
from 0.39 to 0.47 and from 0.89 to 1.76 wt%, respectively,
and are comparable with those obtained by SEM. The mea-
sured Mg/Ca ratios are consistent with the temperature-
dependent solubility of Mg in calcite (Mackenzie et al.,
1983). Sr shows affinity to both aragonite and, to a minor
extent, calcite (Pingitore et al., 1992; Humphrey and Ho-
well, 1999); however, the relatively low Sr contents did
not allow us to attempt a pertinent attribution via Rietveld
refinement, and therefore Sr was assumed to be distributed
in both CaCO3 polymorphs.
Subordinate Fe amounts (up to 0.41% by wt., Tr3),
attributable to either Fe-bearing calcite and/or Fe oxy-
hydroxides, were also detected. Low Al concentrations
(up to 0.23% by wt.) are likely related to the presence of
alumino-silicate minerals (Table 4). Potassium (as K2O),
Na (as Na2O) and P (as P2O5) are 60.5% by wt. In Table 4
the content of selected trace elements (Ba, Cr, Mo, Mn,
Cu, Zn, Cd, Te, As, Sb, Pb, and Th) are also reported.
Mn concentrations are in the range of few hundreds
ppm, showing a significant decrease from the spring to
the pool. Among the other trace elements, Cr, Mo, Cu,
Zn, Cd, Te, Sb, Pb, and Th have lower concentrations,
whereas As and Ba range from 43 to 57 and from 85 to
213 ppm, respectively.
The chemical speciation of S in travertine was investi-
gated by means of DTA. In all the DTA/DTG thermographs
a double endothermic effect appears at peak temperatures of
120 °C. No thermal effects appear on the cooling curves.
As reported in Fig. 7, the calibrated peak temperatures
are 115.5 and 123.9 °C, respectively, and the second one is
rather broader and somewhat less intense. The observed
Table 4
Major (in % by wt.) and trace element (ppm) composition of the travertine deposits collected along the artificial channel at Bullicame 3 after leaching with aqua regia (HNO3:HCl 1:3).
Sample Major elements (wt%) Minor elements (wt%) Total
CaO CaO CO2 CO2 S S MgO MgO SrO SrO FeO Al2O3 K2O Na2O P2O5
(ICP– (SEM) (GC) (SEM)a (GC) (SEM) (ICP–MS) (SEM) (ICP– (SEM) (ICP– (ICP–MS) (ICP– (ICP–MS) (ICP–MS)
MS) MS) MS) MS)

Biotic and inorganic control on travertine deposition

b
Tr1 42.2(1) 53.0(1) 40.3(2) 42.9 1.76(6) 1.7(1) 0.68(5) 1.0(1) 0.398(1) 0.6(2) 0.03(4) 0.06(6) 0.04(4) 0.036(4) 0.02(1) 85.5(5)
Tr2 52.5(1) 53.9(1) 41.9(2) 43.6 1.07(6) 1.2(1) 0.93(5) 1.0(1) 0.470(1) 0.5(2) 0.21(4) 0.13(6) 0.04(4) 0.046(4) 0.03(1) 97.4(5)
Tr3 54.5(1) 53.9(1) 40.2(2) 43.6 1.05(6) 1.0(1) 0.93(5) 1.0(1) 0.432(1) 0.5(2) 0.41(4) 0.23(6) 0.05(4) 0.063(4) 0.01(1) 97.9(5)
Tr4 53.6(1) 54.1(1) 42.2(2) 43.6 1.03(6) 1.0(1) 0.91(5) 0.8(1) 0.457(1) 0.5(2) 0.04(4) 0.06(6) 0.04(4) 0.042(4) 0.02(1) 98.5(5)
Tr5 56.0(1) 53.9(1) 41.8(2) 43.6 1.00(6) 1.1(1) 0.86(5) 0.8(1) 0.447(1) 0.5(2) 0.05(4) 0.04(6) 0.04(4) 0.050(4) 0.02(1) 100.3(5)
Tr6 54.2(1) 53.9(1) 41.5(2) 43.6 1.13(6) 1.0(1) 0.83(5) 0.8(1) 0.459(1) 0.6(2) 0.01(4) 0.02(6) 0.04(4) 0.049(4) 0.02(1) 98.3(5)
Tr7 53.5(1) 54.0(1) 41.4(2) 43.6 0.89(6) 0.9(1) 0.73(5) 0.8(1) 0.425(1) 0.6(2) 0.01(4) 0.02(6) 0.04(4) 0.050(4) 0.01(1) 97.1(5)
Tr8 54.4(1) 53.7(1) 41.4(2) 43.3 1.04(6) 1.0(1) 0.76(5) 0.8(1) 0.435(1) 0.5(2) 0.01(4) 0.04(6) 0.05(4) 0.046(4) 0.01(1) 98.2(5)
Sample Trace elementsc (ppm)
Mo Cu Pb Zn Mn As Th Cd Sb Cr Ba Te
Tr1 0.04(3) 0.35(3) 0.49(3) 1.1(3) 121(3) 85.5(3) 0.3(3) 0.03(3) 0.03(6) 1(2) 43(2) 0.14(6)
Tr2 0.06(3) 1.06(3) 0.71(3) 2.5(3) 168(3) 176.5(3) 0.1(3) 0.08(3) 0.03(6) <1 50(2) 0.19(6)
Tr3 0.13(3) 11.54(3) 2.07(3) 4(3) 184(3) 198.7(3) 0.1(3) 0.18(3) 0.07(6) 5(2) 57(2) 0.15(6)
Tr4 0.04(3) 0.39(3) 0.92(3) 1.5(3) 114(3) 193.8(3) 0.2(3) 0.04(3) 0.03(6) <1 49(2) 0.21(6)
Tr5 0.04(3) 1.66(3) 0.80(3) 0.8(3) 114(3) 213.8(3) <0.1 0.04(3) 0.03(6) 1(2) 52(2) 0.18(6)
Tr6 0.05(3) 0.21(3) 0.44(3) 0.7(3) 107(3) 192.9(3) 0.1(3) 0.11(3) 0.02(6) <1 50(2) 0.18(6)
Tr7 0.01(3) 0.12(3) 0.17(3) 0.4(3) 92(3) 173.4(3) <0.1 0.03(3) <0.02 <1 48(2) 0.17(6)
Tr8 0.03(3) 0.15(3) 0.26(3) 0.6(3) 98(3) 182.9(3) 0.1(3) 0.02(3) <0.02 <1 49(2) 0.14(6)
a
SEM CO2 data were recalculated considering CaO, MgO, and SrO contents as referred to CaCO3, MgCO3, and SrCO3.
b
CaO wt% for sample Tr1 was obtained with AAS.
c
Other trace elements were below the operative detection limit (cf. Acme Analytical Laboratories Ltd.): Hg < 10 ppb; Ag, Au < 40 ppb; Tl < 0.02 ppm; Bi < 0.04 ppm; Co, U, W, Se < 0.2 ppm;
Ni, Ga, Sc < 0.6 ppm; V < 2 ppm; La < 3 ppm; Ti < 10 ppm, B < 50 ppm.

4449
4450 F. Di Benedetto et al. / Geochimica et Cosmochimica Acta 75 (2011) 4441–4455

25.2(1)
28.7(1)
45.1(1)
45.1(1)
43.9(1)
50.8(1)
43.0(1)
47.9(1)
WD
110.4(1)
114.4(1)
111.3(1)
112.2(1)
111.1(1)
111.0(1)
112.0(1)
111.0(1)
EPR parameters (in Gauss, G)

D
25.74(4)
28.19(4)
30.48(4)
30.87(4)
30.14(4)
31.66(4)
30.34(4)
30.97(4)
W6

Fig. 7. Thermograph of the Tr1 sample: calibrated T (in °C, black)

20.74(4)
22.25(4)
21.07(4)
21.40(4)
21.01(4)
20.96(4)
21.35(4)
20.96(4) and DTA (gray) curves are shown. The vertical lines indicate the
maxima of the two thermal effects.
D6
17.032(1)
17.021(1)
17.021(1)
17.022(1)
17.024(1)
17.026(1)
17.031(1)
17.029(1)
c0 (Å)
Theoreticalb

4.9846(1)
4.9827(2)
4.9826(2)
4.9828(2)
4.9832(2)
4.9835(2)
4.9843(2)
4.9840(2)
Structural and EPR parameters of calcite from the travertine deposits collected along the artificial channel at Bullicame 3.

a0 (Å)
365.41(7)
365.47(7)
365.47(7)
365.76(7)
365.63(7)
365.76(7)
366.07(7)
365.59(7)
367.50(5)
V0 (Å3)

367.61

Fig. 8. Lattice constants (in Å) and cell volume (in Å3) for the
investigated samples, from the beginning (Tr1) to the end (Tr8) of
0.2596(5)
0.2599(5)
0.2615(5)
0.2613(5)
0.2677(5)
0.2603(4)
0.2588(5)
0.2631(5)
0.2580(4)
XRPD calcite structural parameters

the artificial channel. Std: synthetic reference sample (Markgraf

0.2567

and Reeder, 1985).

x

temperatures are in agreement with the known values for

17.094(1)
17.086(1)
17.093(2)
17.097(1)
17.094(1)
17.097(1)
17.098(1)
17.088(1)
17.064(1)

thermal effects of elemental crystalline sulfur, the first peak

17.061
c0 (Å)

ascribable to the rhombic–monoclinic transition (113 °C)

and the second to the melting of monoclinic S (123 °C). On
Experimental

the basis of the performed calorimetric calibration, the mea-

Calculated according to the ICP Mg and Sr contents of Table 4.

surement of the area of the melting peak allowed to quanti-

4.9682(2)
4.9699(2)
4.9688(3)
4.9700(2)
4.9697(2)
4.9703(2)
4.9721(2)
4.9703(1)
4.9868(2)

tatively estimate the S-amount present in the sample, that

4.9880
a0 (Å)

resulted in 2.7 wt% in Tr1 sample. Taking into account the

relative uncertainties of the different methods, the obtained
value agrees with the chemical data reported in Table 4.
According to Pokroy et al. (2006), some living organisms
Ref.: Markgraf and Reeder (1985), see text.
XRPD phase composition (wt%)

can control the precipitation of calcite by using S-bearing

Aragonite

macromolecules, the presence of which determines a prefer-

ential route for the growth of bio-calcite. At the end of the
2.6(2)
2.2(2)
2.3(4)
5.6(2)
2.8(2)
2.6(2)
0.9(2)
1.1(2)

process, the macromolecules would remain “embedded” in

the crystallites. On the basis of the present data, the occur-
rence of this second mechanism could not be ruled out,
although it could play a minor role.
Calcite
97.4(5)
97.8(4)
97.7(7)
94.4(5)
97.2(5)
97.4(4)
99.1(4)
98.9(4)

3.4. Structural characterization

The structural data are reported in Table 5. The value of

lattice constants, a0 and c0, refined on the whole series of
travertine samples, are nearly constant; however, they sub-
Table 5

Sample

Ref.a

stantially differ from the literature’s values reported for this

Tr1
Tr2
Tr3
Tr4
Tr5
Tr6
Tr7
Tr8
Std

a
b

range of temperatures (e.g. Reeder, 1983). A standard

 Biotic and inorganic control on travertine deposition 4451

synthetic calcite was analyzed, to verify the correctness of
the acquisition of the XRPD powder patterns and/or the
refinement procedure: the determined lattice constant val-
ues excellently agree with those obtained by Markgraf
and Reeder (1985) on a natural crystal (Table 5).
Bullicame 3 samples show negative Da0 (0.4%) and posi-
tive Dc0 (+0.2%) with respect to the reference values for pure
calcite (Fig. 8). Remarkably, differences in a0 and c0 have
opposite signs. As a result of this lattice variation, an overall
decrease of the cell volume, V0, is observed (Fig. 8). The
refinement of the x fractional coordinate of the oxygen atom,
which is the only free atomic coordinate in the structure, gave
no reliable results. Some variations of this parameter are
observed along the series, but the poor agreement between
literature data and the standard calcite suggests X-ray data
not to be reliable for a more detailed analysis (Table 5).
Theoretical a0 and c0 values, calculated according to the
Mg and Sr contents of each sample (Table 4), are also re-
ported in Table 5. The Ca–Mg substitution (Mg is the most
abundant major ion substituting for Ca, Table 4) is known,
in fact, to induce a decrease of the lattice constants, due to
the difference in the ionic radii between Mg2+ (0.65 Å) and
Ca2+ (0.99 Å). On the other hand the substitution of Ca with
Sr, which was conservatively assumed to be contained in cal-
cite (although in general preferentially partitioned in arago-
nite, Sunagawa et al., 2007), causes an increase of the lattice
constants, Sr2+ (ionic radius: 1.13 Å) being larger than Ca2+
(Pingitore et al., 1992). Thus, both Mg and Sr should be effec-
tive in changing (decrease/increase) both lattice constants,
even if this effect would be more pronounced for c0 (Paquette
and Reeder, 1990). The experimental values for a0 and c0 of
Bullicame 3 travertine are, therefore, apparently due to a
different cause than simple Mg and/or Sr substitution.
and are in agreement with literature values (e.g. Shepherd
and Graham, 1984; Di Benedetto et al., 2005). The experi-
mental D6, D, W6, and WD values are reported in Table 5.
The experimental D values are roughly constant, whereas
W6 and WD significantly change, i.e. they increase as tem-
perature decreases from 57 °C (W6: 25.7 and WD: 25.2) to
47 °C (W6: 30.5 and WD: 45.1). At lower temperatures they
remain almost constant. These data suggest that the mean
strength of the crystal field interaction of Mn(II) does not
change in the whole temperature range, whereas the struc-
tural disorder, i.e. the presence of lattice strains and defects
nearby Mn(II) ions, increases by lowering the temperature
until a steady value is attained. It must be pointed out that
the WD increase with decreasing temperature cannot be ex-
plained in terms of inhomogeneous (dipolar) broadening of
the line, because Mn concentration actually decreases as
temperature decreases (Table 4).
The mean D value, about 111 G, is unexpectedly high
when compared with pure calcite’s value (about 80 G, e.g.
Shepherd and Graham, 1984). This value cannot be ex-
plained even considering the existence of dolomite-like mi-
cro-domains, since Mn(II) in Mg-dolomite sites is reported
to have a D value of the order of 150 G (Shepherd and
Graham, 1984). Moreover, no pre-edge peak characterizing
EPR spectra of Mn(II) in dolomite has been observed at
3100 G (Attanasio, 1999).
No evidence of other paramagnetic impurities, e.g.
Fe(III), of calcite nor other magnetic associated phases,
such as MnO2 or Fe oxy-hydroxides, were observed by
EPR, thus ruling out their possible interference in the
Mn(II) spectral analysis. As a consequence, the relatively
low Fe-content (Table 4) can be attributed to the presence
of the EPR-silent Fe(II) ion in calcite.

3.5. EPR spectroscopy 3.6. Water microorganisms

The experimental EPR spectra show Zeeman and hyper- The main microorganisms isolated along the channel of
fine parameters to remain constant over the whole series Bullicame 3, from high to low water temperatures are

Table 6
Microbial diversity observed in the water samples collected along the artificial channel of Bullicame 3.
Water Fungi Bacteria Microalgae
samples (T)
Tr1 (57 °C) Enterobacteriaceae (Gram bacteria, Cyanobacteria: Synechococcus eximius,
oxidase ) Chroococcus cf. yellowstonensis
Tr3 (47 °C) Corynebacteriaceae (Gram+ bacteria) Cyanobacteria: Synechococcus eximius,
Enterobacteriaceae (Gram bacteria, Lygnbia limnetica, Phormidium sp.
oxidase )
Tr5 (37 °C) Corynebacteriaceae (Gram+ bacteria) Cyanobacteria: Synechococcus eximius, Oscillatoria sp.
Enterobacteriaceae (Gram bacteria, Bacillariophyta: Navicula sp.
oxidase ) Chlorophyta: Dyctiosphaerium sp.,
Pseudomonadaceae (Gram bacteria, Scenedesmus acuminata
oxidase +)
Tr7 (27 °C) Sporidiobolaceae: Cyanobacteria: Nostoc comune, Anabaena circinalis,
Rhodotorula sp. Oscillatoria sp.
Bacillariophyta: Navicula radiosa, Pinnularia sp.,
Gyrosigma alternatum
Chlorophyta: Chlorella sp., Chlamidomonas sp.,
Pediastrum cf. biradiatum, Scenedesmus acuminata
Euglenaceae: Phacus sp.
4452 F. Di Benedetto et al. / Geochimica et Cosmochimica Acta 75 (2011) 4441–4455
Enterobacteriaceae and Microalgae at 57 °C, Corynebacte-
riaceae, Enterobacteriaceae, and Microalgae at 47 °C;
Corynebacteriaceae, Enterobacteriaceae, Pseudomonada-
ceae, and Microalgae at 37 °C and Sporidiobolaceae (Rho-
dotorula sp.) and Microalgae at 27 °C (Table 6). A relatively
high biodiversity of Microalgae (Cyanobacteria, Bacilla-
riophyta, Chlorophyceae, Euglenacea) was observed in
the water samples at 27 °C, whereas at higher temperature
Cyanobacteria and Enterobacteriaceae are dominating. Set-
ting aside the Rhodotorula sp. pigmented yeast found at
27 °C, no fungal diversity was identified (Table 6).
4. DISCUSSION
4.1. Isotopic fractionation among gas, liquid, and solid phases
A relevant difference is observed between the experimen-
tal isotopic d18O and d13C values in the system: the former
values appear consistent with a balanced equilibrium tem-
perature-dependent isotopic exchange between the gas
and the liquid phase (Scholz et al., 2009), whereas the latter
values mark a solid phase clearly far from the equilibrium
with the contact liquid phase (Skidmore et al., 2004). Thus,
experimental data would apparently indicate that isotopic
fractionation of C and O follows different pathways.
The observed d13C values for travertine samples can be
in principle attributed to either a kinetically controlled inor-
ganic deposition process or a bio-assisted precipitation of
calcite. According to the first hypothesis, the geometrical
dimensions of the spring–channel system would not allow
a complete T-dependent re-equilibration of the chemical
species in the whole volume of water, at the given boundary
conditions (flux of the water, time scales of degassing and
precipitation processes, time scale of the diffusion processes
involving the considered species) (Skidmore et al., 2004;
Scholz et al., 2009). According to the second hypothesis,
during bio-mineralization isotopic fractionation processes
caused by bio-mediated precipitation would produce 12C
enrichment in carbonates (e.g. Guo et al., 1996; Zhang
et al., 2004). Several authors attributed out-of-equilibrium
isotopic signatures of travertines from many sites to this
process (Folk, 1992, 1993a,b, 1994, 1997; Pentecost, 1995;
Vasconcelos et al., 1995; Chafetz and Guidry, 1999; Kirk-
land et al., 1999) including some sites from the Viterbo
Area (Chafetz and Lawrence, 1994; Allen et al., 2000).
The simulation model presented in this study calculates
the evolution of the chemical species distribution in the sys-
tem in absence of biotic effects. The agreement between sim-
ulated and experimental isotopic fractionation values at
both the gas/liquid and liquid/solid interfaces (Figs. 4a
and 5) suggests that the simple diffusion-controlled kinetics
of transport and distribution of the chemical species could
justify a precipitation process of travertine with an anoma-
lous isotopic signature. However, the simultaneous occur-
rence of bio-assisted precipitation processes cannot be
ruled out. In our opinion, no further reliable indications
can be extracted from our analysis concerning isotopic sig-
nature, and discrimination between the two hypotheses is
not allowed, as already pointed out by Skidmore et al.
(2004).
4.2. Structural and EPR features
The travertine samples are characterized by a double
evidence of unexpected structural features: both XRPD
and EPR, in fact, exhibit anomalous values of the lattice
and the crystal field interaction, respectively.
The observed modified lattice of Bullicame 3 travertine
cannot be simply explained in terms of its elemental chem-
istry. In fact, the substitution of Mg and Sr for Ca in the
calcite lattice is expected to induce opposite volume effects
and cannot give rise to the observed variation of the lattice
constants with a negative value for Da0 and a positive one
for Dc0. On the other hand, no other minor/trace compo-
nent, apart from Mg and Sr, is abundant enough to play
a significant role in the production of lattice variations.
Bischoff et al. (1983), Paquette and Reeder (1990), and,
more recently, Pokroy et al. (2006) point out that the c0/a0
ratio can be a useful parameter to indicate the presence of
bio-mineralizing activity: bio-mineralized calcite is indeed
always characterized by c0/a0 ratios higher than those re-
ported as a function of the Mg content. Bischoff et al.
(1983) suggest a c0/a0 ratio of 3.437, particularly close to
the value determined in the present work (3.439).
In the EPR spectrum of Mn(II), as in general for transi-
tion metal ions, ZFS values are directly related to the chem-
ical and structural environment of the paramagnetic center.
In Mn(II), hyperfine and Zeeman anisotropies are actually
negligible compared to the crystal field. In calcite, the Ca
site is a trigonally distorted octahedron, with D3d point
symmetry. The Mn(II) replacing Ca(II) gives rise only to
minor local distortions (Cheng et al., 2001), inducing an ax-
ial zero-field splitting. The anisotropy of this interaction,
parameterized by the D term, increases by increasing the de-
gree of distortion of the polyhedron.
The observed D values, significantly larger than those re-
ported in literature for pure calcite (e.g. Wildeman, 1970;
Angus et al., 1979) indicate a strong distortion of the geom-
etry around the Mn(II) ion. The observed shrinking of the
unit cell evidenced by X-ray data was tentatively associated
to the observed ZFS values. However, the structural
changes of the Ca octahedra are too small to induce the ob-
served relevant increase (+39%). For comparison, one has
to consider the D value observed for Mg-sites in dolomite
(about 150 G; Shepherd and Graham, 1984) that is caused
by a reduction of the cation–oxygen distance of 12%: here
the distance decrease is about 1%. Thus, the ZFS values ob-
served in this work cannot be attributed either to Mn in a
distorted Ca site or to a Mn in dolomite-like sites. Accord-
ing to Kretz (1982), the tendency of Mn(II) to be systemat-
ically related either to Ca- or to Mg-sites in Mg-bearing
calcites or dolomites reflects the conditions of rock forma-
tion. In the case under study, it is therefore conceivable that
the large D parameter has to be related to the proximity of
Mn(II) ions to highly distorted Mg containing sites. The ab-
sence of any dependence on Mn(II) concentration (Table 4),
temperature and pH, suggests that the co-precipitation of
Mn(II) with calcite does not follow a random path; a given
association between Mn and Mg is retained in the whole
series of travertine encrustations deposited from the source
down to the pool.
 Biotic and inorganic control on travertine deposition
The circumstantial evidence of a non-random clustering
of Mn and Mg is reinforced by the trend observed in the
WD parameter, related to the spread of the ZFS interaction
and to the lattice chemical/structural disorder. Indeed, in a
random process this parameter should decrease with Mn(II)
concentration, since the probability of finding Mn in non-
equivalent structural/chemical environments is a function
of the Mn content (e.g. Angus et al., 1979; Kralj et al.,
2004). In the Bullicame 3 samples, the WD behavior indi-
cates an increasing local disorder as temperature decreases,
in spite of the sharp decrease of the Mn concentration along
the channel. This supports the idea of non-random process
acting in the precipitation, which in turn is a clue of a bio-
assisted precipitation.
4.3. Model of the spring system and of travertine deposition
On the basis of the complete set of experimental data,
the Bullicame 3 system, constituted by the thermal spring,
the channel and the final pool, can be considered formally
divided into two sections, each having peculiar composi-
tional and isotopic features.
Deep hot water, enriched in gas, is taken up to the sur-
face of the spring, and subsequently flows down to the pool
at a constant rate and with decreasing temperature. Along
the channel, partial H2O as well as excess CO2 evaporation
occurs. As a consequence, pH increases whereas HCO3 de-
creases. This process is tracked by water and gas chemistry,
and can be described as a fast process. The isotope fraction-
ation follows the fast exchange at the water/atmosphere
interface, and the isotopic anomaly of the spring is progres-
sively shifted towards values typical of meteoric water. The
HCO3–CO2 isotopic equilibrium rapidly attained at each
temperature can be conveniently modeled through isotopic
fractionation calculations. Simultaneously, precipitation of
travertine occurs at the water/solid interface, where experi-
mental data appear to be more ambiguous: the isotopic
fractionation, the calcite structural features, and the spec-
troscopic data clearly indicate an apparent non-equilibrium
process. However, even if the isotopic signatures of traver-
tine are assumed to depend on precipitation under a kinet-
ically-controlled regime of transport, the same cannot be
said regarding structural and spectroscopic evidence. This
fact deserves, in our opinion, further consideration.
ZFS indicates preferential clustering pathways for Mg
and Mn that cannot be attributed to a pure stochastic pro-
cess. On the other hand, structural XRPD analysis of the
calcite fraction clearly indicates a bio-mineralizing process
as a possible candidate to explain all the observed anoma-
lies: the lattice distortion can indeed be considered a strong
indication for such explanation.
Furthermore, the presence of the Sinechococcus cyano-
bacteria in three out of the four analyzed water samples
supports the idea that the travertine deposit, mantling the
artificial channel of the Bullicame 3 thermal spring dis-
charge, likely exhibits some bio-assisted precipitation, as
these bacteria are known to participate in the formation
of biogenically-precipitated calcite (Lee et al., 2006).
The structural deformation of the calcite lattice would
be produced by protein activity of bio-precipitation
4453
(Pokroy et al., 2006). According to inorganic mechanisms,
Mg and Mn ions, less accepted in the Ca-carbonate bacte-
rial metabolism, would probably co-precipitate with
CaCO3., but it is noteworthy to mention that Pokroy
et al. (2006) do not exclude the fixing of Mg through bacte-
rial activity. Finally, the intracellular bacterial activity
would yield a constant precipitate masking the external
physico-chemical input. Indeed, Pokroy et al. (2006) stress
that sulfur anomalies associated to the shrinking of the cal-
cite unit cell may be attributed to “biologic S”, thus linking
the two experimental observations as effects of a unique
bio-mediated precipitation process. As previously noted,
also in the Bullicame 3 samples a sulfur anomaly is
occurring.
5. CONCLUSIONS
According to the results presented in this study, we can
describe the Bullicame 3 system (hot spring + artificial
channel + pool) as constituted by two markedly different
subsystems:
– water/gas interface, where a fast exchange between the
solute and the dissolved gas species allows the system to
achieve chemical and isotopic equilibrium as the water
flows along the system, while temperature decreases;
– solid/water interface, where fast travertine precipitation
occurs; carbon isotopic ratios in the solid phase, suggest
non-equilibrium conditions, while chemical, structural
and spectroscopic data point to a bio-assisted process.
This conclusion is also strongly supported by the identi-
fication of calcifying cyanobacteria throughout the
channel.
The fact that an active role played by bacteria is proba-
bly operating in a high temperature spring system, as Bulli-
came 3, opens interesting perspectives for the study of more
active systems with similar characteristics, whose composi-
tional and isotopic profiles could be properly analyzed in
the framework of the theoretical modeling herewith pre-
sented. One of the most relevant aspect of this characteriza-
tion, i.e. the anomalous ratio between the lattice constants,
could be used as a proxy for microbiological activity in cal-
cite-bearing geomaterials.
The approach presented in this work combines isotopic,
compositional, structural and EPR spectroscopic investiga-
tions. The results obtained in this study suggest that this ap-
proach can be profitably applied to the study of other
systems, as fossil travertine deposits, where the absence of
the coexisting parent waters prevents the direct knowledge
of the microbiological environment active during the depo-
sition. On this basis, future applications on fossil travertine
will be relevant in order to reconstruct the environmental
(and potentially paleoclimatic) conditions existing during
the deposition.
ACKNOWLEDGMENTS
The manuscript draft benefited of the critical reading by E.
Valsami-Jones (Imperial College, London), G. Lampronti (Univer-
sity of Bologna), F. Pineider and G.P. Bernardini (University of
4454 F. Di Benedetto et al. / Geochimica et Cosmochimica Acta 75 (2011) 4441–4455
Florence), to whom the authors wish to express their warmest grat-
itude. The authors acknowledge also Filippo and Claudio Di
Benedetto, who provided the historical information about the site,
and P. Costagliola (University of Florence) for the useful discus-
sions about the structural remnants of microbiological activity.
Miria Borgheresi is acknowledged for her help in the sampling
campaign. FDB benefited of a research grant funded by the Regio-
nal Administration of Tuscany. Luca Pardi and Giordano Monteg-
rossi thank the Italian National Research Council (CNR) for
support. Authors wish to express their gratitude to the anonymous
referees, for their stimulating criticisms, which largely improved the
text.
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