THERMAL AND CHEMICAL STABILITY OF THE SOLID STATE RNA SAMPLES:
COULD IT SURVIVE AT THE EXTREME CONDITIONS OF THE EARLY EARTH?
1

Gradova M.A.1, @, Gradov O.V.1, Naganovskiy Yu.K.2, Sergeev A.I.1

Semenov Federal Research Center for Chemical Physics of the Russian Academy of Sciences,
119991, Russian Federation, Moscow, Kosygin Street, 42All-Russian Research Institute for Fire Protection of Ministry of Russian Federation for Civil
Defense, Emergencies and Elimination of Consequences of Natural Disasters, 143903, RussianFederation, Moscow Region, Balashikha, mkr. VNIIPO, 12
e-mail: m.a.gradova@gmail.com(Received: August, 9, 2020 Accepted: November, 5, 2020)

Abstract
In this work physical and chemical properties of RNA
and its sodium salt (Na-RNA) have been studied at
extreme conditions similar to those abundant at theprebiotic stage of chemical evolution. Characterizationof dry RNA samples has been performed by thermal
analysis (TGA and DTA) and NMR relaxation methods.
Aqueous solutions of the above samples have been
additionally studied using UV-Vis spectroscopy anddynamic light scattering. It has been shown that themechanism and rate of thermal destruction, as well as
the hydration degree of the samples significantly differ
for the two forms of RNA studied. A chemical
modification of the solid state RNA samples uponstorage includes both oxidation of the nitrogenous
bases and hydrolysis of phosphodiester bonds leading
to the changes in the absorption spectra and different
solubility of the RNA and Na-RNA samples.

validation of the numerous scenarios of abiogenesis andmolecular evolution should first of all include aphysicochemical analysis [5] using the phase diagramof the corresponding systems.
Transition from abiogenic to prebiological systems isa set of phase transitions [6-13], and the resulting
system is a multiphase system that is stable in abiologically relevant range of terrestrial environmental
conditions. If the system is not optimal for a given
environment, it does not survive due to thephysicochemical natural selection [14,15]. One of themain criteria for the successful transition from chemical
to biochemical evolution is reproduction or replicationof the genetic code carrier [16,17]. Hence, all the
scenarios proposed must ensure the safety of the carrier(or carriers [18,19]) at all stages of molecular evolution.
If a certain scenario of molecular evolution includes an
episode of disruption of carriers of genetic information,
further development of life based on this substance is
impossible. Hence, it is necessary to analyze thermal
stability of nucleic acids under all temperature
conditions in which DNA / RNA could appear duringmolecular evolution [20,21].
To date, there are several hypotheses linking thedevelopment of the ability to replicate, as well as theaccumulation of mutations in DNA / RNA, with thecyclic temperature changes in the medium, which could
lead to thermal cycling of the code carriers by analogy
with the known polymerase chain reaction (PCR)
protocols [22-24]. Beyond the temperature range
suitable for PCR, this principle is not applicable, since
thermodynamics of irreversible processes leads to thefact that the system, brought too far from theequilibrium, not only fails to produce its own copies,
but also can not more exist in its initial state, going intoanother phase field [25-27].
Nowadays there are modern techniques combining
thermal cycling of nucleic acids in PCR and their
thermal analysis [28], but they operate within a narrowtemperature range in which the state of DNA and RNA
is well established. Meanwhile, in many scenarios of
the origin of life temperature significantly exceeds the
PCR threshold less than 100ºC. For example, theso-called “Hot Earth” hypotheses consider temperatures
much more than 100ºC, in a deep-water hydrothermal

Keywords: ribonucleic acid, sodium ribonucleinate,
themogravimetric analysis, differential thermal analysis,
NMR relaxation.
Introduction
The main problem in verification of numerous
scenarios of abiogenesis and chemical / prebiotic
evolution under the certain astrochemical andgeochemical conditions is their consistency with theminimal physicochemical requirements to theconditions of existence (structure preservation andsustainable functioning) for the crucial substances
involved in the above scenarios. In other words, at thegiven natural temperatures and pressures in the phase
diagram a possible prebiotic system should have thesame phase composition and functionality as its modern
biologically active analog or evolutionary “descendant”.
However, in the high temperature range the system will
decompose while in the ultra low temperature range
components of the modern cytoplasm crystallize (which
is not always reversible [1] and often disrupts theirconformation [2,3] and biological activity [4]). In
multicomponent microheterogeneous systems likecytoplasm one component may start to decompose
before another one, therefore, the loss of functionalityof the whole system will be determined not by theentire composition of the system, but by the limiting
component, according to the Liebig principle. Thus,

Viva Origino 2020, 48, 1© 2020 by SSOEL Japan1

Viva Origino 2020, 48, 1

origin of life concept (which also implies high pressure
and supercritical fluids [29]) the temperature reaches
150–200ºC, in volcanic systems the temperature is
above 400–800ºC [30-32], and finally in panspermia
speculations the rock passing through the dense layersof the atmosphere can be exposed to the temperature
difference of n*103 ºC and heating of the meteorite /micrometeorite surface can exceed n*103 ºC [33-35].
Therefore, an extended thermal analysis of RNA from25 to 800ºC is required. Since in real conditions thenucleic acid sample heating is always accompanied by
the loss of mass, it is possible to apply
thermogravimetry. In order to simulate an anoxigenic
atmosphere of the early Earth [36], the experiments canbe carried out in nitrogen atmosphere.
The vast majority of works on thermal analysis of
nucleic acids deals with DNA as the main carrier ofgenetic information in modern biological systems[37-39] which possesses higher thermal and chemical
stability (compared to RNA) as a result of natural
selection during chemical evolution. However, thewidely recognized concept of the RNA-world
postulates the primacy of RNA as the earliest carrier ofthe genetic code [40,41]. In this regard, in order toverify the concepts of abiogenesis suggesting theleading role of RNA at the prebiotic stage of chemical
evolution, it is necessary to address physical andchemical properties of RNA in extreme conditions, inparticular, at dehydrated state, upon storage and underhigh temperatures abundant for the geochemical
conditions of the emergence and development of life on
early Earth.

0.005 mg). Thermoanalytical measurements werecarried out in an inert (nitrogen) atmosphere with theheating rate of 20°C / min. The following
characteristics
were
calculatedfromthethermoanalytical curves (TG and DTG): temperature
ranges of destruction, mass loss within the temperature
ranges (%), DTG maximum temperature (ºС), mass loss
rate (% / min), chark oxidation rate (% / min).
Molecular dynamics of the most relevant samples 3,
5, 8 and 9 was studied by proton magnetic relaxation
using a Minispec PC-120 NMR relaxometer (Bruker,
Germany) with an operating frequency of 20 MHz.
RNA and Na-RNA powders were placed into a specific
NMR tube with a diameter of 5 mm and hermeticallysealed. All the measurements were carried out at 25 ±0.5ºC. To estimate the spin-spin relaxation time T2 andthe fraction of protons with different mobility we used
the techniques for studying the free induction decay(FID) of the signal after a 90°-pulse and the pulse
sequence CPMG [42,43]. Typically, FID is used toevaluate proton mobility in condensed systems,
including solid-state polymers, with the relaxation
times up to microseconds and the decay time of the freeinduction signal about 100 µs. When the signal decay islonger than 100 µs, distortions are introduced into themeasurements due to the inhomogeneity of themagnetic field. CPMG method removes the influenceof magnetic field inhomogeneity, but the measurements
can be started only from the time of about 70 µs, i.e. thecharacteristics of the mobile proton fraction aremeasured, while the study of the rapidly decaying partof the curve is difficult. In this work, the FID signal has
been studied from 10 µs up to 100 µs with the sampling
frequency of 1 MHz (90°-pulse duration was 2.7 µs,
dead time - 7 µs, the number of repetitions - 100 andthe delay between the subsequent pulses was 2 s). Т1
time for all the samples was less than 0.3 s. The time
range of the measured curve in CPMG method was
1.5–2 ms. The time between 90°- and 180°-pulses τ =20 µs, the number of repetitions was also 100 and thetime between two pulse sequences was 2 s.
Magnetization decay curves were calculated using
Origin 6.1 software.
Particle size distribution in RNA (sample 9) and
Na-RNA (sample 3) aqueous solutions (C = 10 mg/ml)
was estimated using dynamic light scattering method
on Zetasizer ZS (Malvern Instruments, USA)
instrument. Excitation and emission spectra of theabove solutions were recorded using luminescence
spectrometer Perkin Elmer LS-50 (Perkin Elmer, USA)at room temperature in 10 mm quartz cells within the
wavelength range of 250–800 nm with a resolution of
0.5 nm. Data processing was performed using Origin
6.1 software.

Experimental
Crystalline powders of yeast ribonucleic acid (RNA,sample 9) and its sodium salt (Na-RNA, samples 1-8)
were purchased from Reachem (Moscow, Russia) andused as received. Both reagents were of chemical grade
and contained low-molecular fractions of the yeast
RNA extracts. Solid state RNA and Na-RNA samples
have been studied by TGA and DTA using a standard
test method ASTME 1131-08 for compositional
analysis by thermogravimetry. Characteristics ofthermal destruction of materials (10–20 mg) werestudied using an automated modular thermal analysis
system "Du Pont 9900". Software allowed to control
the operation of thermoanalytical modules, to monitor
the measurement process in real time, to register the
sample mass change (TGA) in the form of data files
and to process the results obtained. Processing andanalysis of the thermoanalytical curves obtained was
carried out using «File Modification V 1.0» andUniversal Analysis 2000 software. Thermal balances
TGA-951 were calibrated using reference substances
recommended by the International Committee for
Thermal Analysis (ICTA) and a gold reference (50 ±2Viva Origino 2020, 48, 1

Results
The data obtained reveals a similar thermal
decomposition pathway for all the Na-RNA samples(1-8) and a significantly different behavior for pure
RNA sample (9) (Fig. 1a). Below 200ºC the yield of
volatiles is 15–20 mass % with the mass loss rate of
3.6–4.6 % per minute. Between 200 and 500ºC thermal
destruction of the samples occurs with the mass loss of
40–50% and the mass loss rate of 6.5-9% per minutewith the temperature maximum of 295-300ºC. Above
500ºC a very low rate of pyrolysis (1.2–0.2% / min) is
observed. For pure RNA sample (9) in the temperature
range below 500ºC there are three distinct stages ofthermal destruction with the maxima at 90ºC, 225ºC
and 300ºC, and the corresponding thermal
decomposition rates of 1.2, 2.8 and 4.5% per minute.
Above 500ºC there is also a very low pyrolysis rate of
1.2–0.3% per minute. After changing the inert N2
atmosphere to air, the carbonized fraction which differs
in different samples undergoes oxidation with different
rates (Fig. 1b). According to DTG curves, a different
nature of the carbonized sample oxidation is observed
at 800ºC.
The difference in thermal decomposition observed
between RNA and Na-RNA samples in a solid state has
been proved by NMR relaxation data. The measured
FID signal can be described by three components: thefirst fast-decaying signal from the protons in the “solid”
dry phase (CH, CH2, NH and several OH groups of thecrystal and amorphous parts of the sample) with thecorresponding spin-spin relaxation time T21, and two
slower decaying components (they can include water
protons and protons of the RNA structure in OH, NH
groups exchanging with it) with the corresponding
spin-spin relaxation times T22, T23. The results obtainedusing FID methods are summarized in table 1.
From the data obtained it can be concluded thatsample 9 demonstrates relaxation close to a solid statewith the significantly limited proton mobility, while theother samples 3, 5 and 8 contain two types of protons,
including low-mobile ones (predominant in sample 3)
and more mobile ones, predominant in samples 5 and 8,
which can be attributed to the higher water content in
the latter samples, resulting in plasticizing effect ofwater on biopolymers (Fig. 2), which is consistent withthe above discussed TGA data.
Additional experiments with the aqueous solutions of
the stored RNA and Na-RNA samples revealed asignificant difference in their solubility with the
Na-RNA samples 1-8 readily dissolving intotransparent molecular solutions with pH 6.5 and RNA
sample 9 forming a turbid colloidal suspension with pH

Figure 1. a - TG (1) and DTG (2) curves of the initial
RNA and Na-RNA samples; b - TG (1) and DTG (2)curves of carbonized sample oxidation at 800°C.

Table 1. Fast and slow FID components and their percentage ratio in RNA samples.
Samplenumber

Т21,
µs

%

Т22,
µs

%

Т23,
µs

%

3

10

90

22

6

350

4

5

10.7

75

60.2

8

430

17

8

10.2

74

23

14

350

12

9

11

100

3

Viva Origino 2020, 48, 1

products of the nitrogenous bases, produced upon the
RNA sample storage at ambient conditions inoxygen-containing atmosphere without any specificprotection. This process can be facilitated by the sample
exposure to the UV light, high temperature, or
treatment by the reactive oxygen species [44,45]. The
primary product of RNA oxidation, according to theliterature available, is 8-hydroxyguanosine [46,47].
Further chemical transformations can lead to theformation of a complex mixture of purine derivatives[48]. It is also noteworthy that single-stranded RNA
without hydrogen bonds protecting its nitrogenous
bases is known to be more susceptible to oxidative
damage compared to the double-stranded DNA [49,50].
Oxidative damage of the RNA structure results in thealteration of the normal base pairing, and hence,
prevents RNA molecules from correct operation duringtranscription and translation processes [51].

Figure 2. Free induction decay signal, sampled with anacquisition time 1 µs.

3.5 at the same concentration of 10 mg/ml. According
to DLS measurements, this suspension demonstrates awide particle size distribution with the averagehydrodynamic diameter of the molecular aggregates
being about 200 nm, while the mean particle size in aless polydisperse molecular solution of sample 3 isabout 2 nm (Fig. 3). This difference can be attributed tothe partial hydrolysis of RNA with the subsequentacidification of the solution preventing the polymer
from dissociation. Upon addition of NaOH,
neutralization results in ionization of the phosphategroups leading to the increased solubility of thepolyanion and the transition from a globular to a linear
macromolecule conformation.

Figure 4. Excitation and emission spectra of
Na-RNA solution (sample 3).

Discussion
Thus, there are at least three types of abiogenetic
conditions that impose temperature restrictions on thepossibility of RNA participation in prebiologicalprocesses. Firstly, it is not possible to consider the“RNA world” operating under hydrothermal conditionsat temperatures of about 400°C (within the frameworkof the so-called “hot scenario”). As it is correctly notedin [52], "one may deny the possibility of the existenceof the RNA world under hydrothermal environments on
the basis of the qualitative understanding that RNA
should normally be weak at high temperatures". The
above cited paper shows that approaching a temperatureof 200–300ºС leads to the RNA degradation, since themaximum temperature for maintaining its functionality
is close to the survival temperatures ofhyperthermophilic organisms (100–120ºС).
Secondly, despite the well-known concepts on thepossible presence of RNA in meteorites and cosmic
dust involved in the panspermia hypothesis (pre-RNA

Figure 3. Particle size distribution for Na-RNA (a) andRNA (b) solutions.
For all the samples studied regardless of the chemical
form of RNA pronounced spectral changes were also
observed upon storage. In addition to the characteristic
UV absorption maxima at 230, 260 and 280 nm typicalfor RNA molecules, a clear bathochromically shifted
absorption band was observed at 335 nm with thecorresponding fluorescence emission band at 415 nm.
Such new bands can be attributed to the oxidation

4

Viva Origino 2020, 48, 1

world [53,54]), it is difficult to believe in the safety of
RNA in such objects and the possibility of its safedelivery to the Earth in this way. The inevitable RNA
decomposition at temperatures above 300ºС will notallow it to reach the planet surface even in the absenceof modern dense layers of the Earth atmosphere. Evenless plausible is the hypothesis of the presence of RNA
on the surfaces of other astrobiological objects, forexample, on Venus, where the surface temperaturereaches 280°C.
Variations in the concept of the RNA world localized
within the aquatic microreservoirs in volcanic aquifers[55], as well as hypotheses appealing to the effect oflightning in volcanic conditions, also contradict the
known data on the temperature in such systems, atwhich the lifetime of such aquatic microreservoirs
could not be sufficient enough for proceeding ofmolecular / chemical evolution or selection ofmolecular quasispecies in the RNA world [56-58]. The
only exceptions to this rule are marine underwater
volcanoes: the extremophile hyperthermophiles foundin them can live at temperatures of the order of 100°C[59], which, however, is many times lower thanstandard volcanic temperatures. It is known that a hotcore of the volcanic plume has temperature above
600°C, while magmatic gas - up to 1000°C [60]. Thus,in the “volcanic” versions of the RNA world,
thermobarogeochemistry of these systems contradicts
the structural chemistry of nucleic acids, which makes
impossible molecular / chemical evolution in
populations of RNA molecules in volcanic
communities.

References
1. Kurganov, B. I., Lyubarev, A. E.,
Sanchez-Ruiz, J. M., Shnyrov, V. L.
Biophysical chemistry 69, 125 (1997)
2. Sung, W., Jeon, J. H. Physical Review E 69,
031902 (2004)
3. Goldstein,R.
N.,
Stefanovic,
S.,
Kallenbach, N. R. Journal of Molecular
Biology 69, 217 (1972)
4. Protasevich, I., Ranjbar, B., Lobachov, V.,
Makarov, A., Gilli, R., Briand, C., Lafitte, D.,
Haiech, J. Biochemistry 36, 2017 (1997)
5. Budin, I., Szostak, J. W. Annual Review ofBiophysics 39, 245 (2010)
6. Mathis, C., Bhattacharya, T., Walker, S.
I. Astrobiology 17, 266 (2017)
7. Uversky, V. N. Advances in Colloid andInterface Science 239, 97 (2017)
8. Ferreira, C. P., Fontanari, J. F. PhysicalReview E 65, 021902 (2002)
9. Sleutel, M., Lutsko, J., Van Driessche, A. E.,
Durán-Olivencia, M. A., Maes, D. Nature
Communications 5, 1 (2014)
10. Hyman, A. A., Simons, K. Science 337,
1047(2012)
11. Sardanyés, J., Solé, R. V. Journal of
Theoretical Biology 243, 468 (2006)
12. Hansma, H. G. Biophysical Journal 114, 440a(2018)
13. Kompanichenko,
V.
N. Frontier
Perspectives 13, 22 (2004)
14. Fry, I. Origins of Life and Evolution of
Biospheres 41, 3 (2011)
15. Morcos, F., Schafer, N. P., Cheng, R. R.,
Onuchic, J. N., Wolynes, P. G., Proceedings of
the National Academy of Sciences 111, 12408(2014)
16. Pross, A. Origins of Life and Evolution of the
Biosphere 34, 307 (2004)
17. Orgel, L. E., Lohrmann, R. Accounts ofChemical Research 7, 368 (1974)
18. José, M. V., Morgado, E. R., Govezensky, T.,
Aguilar, I. Journal of Cosmology 5, 854(2010)
19. Levy, M., Miller, S. L. Proceedings of the
National Academy of Sciences 95, 7933 (1998)
20. Nakano, S. I., Kanzaki, T., Sugimoto,
N. Journal of the American Chemical
Society 126, 1088 (2004)
21. Davanloo, P., Sprinzl, M., Watanabe, K.,
Albani, M., Kersten, H. Nucleic Acids
Research 6, 1571 (1979)
22. Varfolomeev, S. D., Tsybenova, S. B., Bykov,
V. I., Goldberg, V. M. Doklady Physical
Chemistry 481, 117 (2018)23. Varfolomeev,
S.
D. Mendeleev
Communications 17, 7 (2007)
24. Varfolomeev, S. D., Lushchekina, S. V.
Geochemistry International 52, 1197 (2014)
25. Wereszczynski, J., Andricioaei, I. Proceedingsof the National Academy of Sciences 103,

Conclusions
The performed experiments revealed a significant
difference in the thermal decomposition behavior andhydration degree of the solid state samples ofribonucleic acid and its sodium salt, as well as theirsolubility difference upon storage due to the chemicaltransformation of the samples. According to the TGA
data, the first structural transformation for the RNA
samples occurs at 70°C and a complete decomposition
is observed at 300°C, which prevents from speculations
on the possibility of RNA participation in the prebiotic
chemical reactions at higher temperatures.
Acknowledgments
The authors gratefully acknowledge Dr. Bychkova A.V.
(N.M. Emanuel Institute of Biochemical Physics RAS)
for performance of the DLS measurements andAlexandrov P.L. (Shemyakin-Ovchinnikov Institute ofBioorganic Chemistry, RAS) for the technicalassistance. The work was carried out in the frameworkof the State Assignment of Ministry of Science andHigher Education of the Russian Federation (project0082–2018–0006,
registrationcodeАААА-А18-118020890097-1).

5

Viva Origino 2020, 48, 1

16200 (2006)
26. Woo, H. J., Wallqvist, A. Physical Review
Letters 106, 060601 (2011)27. Yang, H., Zhuo, Y., Wu, X. Journal of Physics
A 27, 6147 (1994)
28. Dinges, W. L. U.S. Patent No. 8 153 374 (Apr.
10, 2012)
29. Budisa, N., Schulze-Makuch, D. Life 4, 331(2014)
30. Huber, C., Wächtershäuser, G. Science 314,
630 (2006)
31. Huber, C., Eisenreic, W., Wächtershäuser,
G. Tetrahedron Letters 51, 1069 (2010)
32. Russell, M. J., Hall, A. J., Cairns-Smith, A. G.,
Braterman, P. S. Nature 336, 117 (1988)
33. Melcher, C. L. Geochimica et Cosmochimica
Acta 45, 615 (1981)
34. Kosarev, I. B. High Temperature 47, 777(2009)
35. Qu, F., Chen, J., Sun, D., Bai, J., Zuo, G.
Aerospace Science and Technology, 86, 659(2019)
36. Shaw, G. H. Geochemistry 68, 235 (2008)
37. Hoyer, H. W. , Barrett, E. J. Analytical
Biochemistry 17, 344 (1966)
38. Inaba, S., Hosoe, Y., Sasaki, Y. C., Sekiguchi,
H., Oda, M. Biophysical Journal 116, 191a
(2019)
39. Koppert, J., Jean-Ruel, H., O’Neill, D., Harder,C., Willmore, W., Ianoul, A., Albert, J.
Analytical and Bioanalytical Chemistry 411,
6813 (2019)40. Orgel, L. E. Critical Reviews in Biochemistry
and Molecular Biology 39, 99 (2004)
41. Spirin, A. S. Molecular Biology 39, 466(2005)
42. Carr, H.Y., Purcell, E.M. Physical Review 94,
630 (1954)
43. Meiboom, S., Gill, D. Review of Scientific
Instruments 29, 688 (1958)
44. Liu, Z., Chen, X., Li, Z., Ye, W., Ding, H., Li,
P., Aung, L. H. H. International Journal of
Molecular Sciences 21, 5022 (2020)
45. Li, Z., Malla, S., Shin, B., Li, J. M. Wiley

Interdisciplinary Reviews: RNA 5, 335 (2014)
46. Kong, Q., Lin, C. L. G. Cellular andMolecular Life Sciences 67, 1817 (2010)
47. Wurtmann, E. J., Wolin, S. L. Critical Reviewsin Biochemistry and Molecular Biology 44, 34(2009)
48. Kuznetsova, A. A., Knorre, D. G., Fedorova,
O. S. Russian Chemical Reviews 78, 659(2009)
49. Li, Z., Wu, J., DeLeo, C. J. IUBMB Life 58,
581 (2006)
50. Li, Z., Chen, X., Liu, Z., Ye, W., Li, L., Qian,
L., Ding, H., Li, P., Aung, L. H. H. Frontiersin Molecular Biosciences 7, 184 (2020)
51. Ishii, T., Sekiguchi, M. DNA Repair 81,
102666 (2019)
52. Kawamura, K., Da Silva, L., Ogawa, M.,
Konagaya, N., Maurel, M. C. BIO Web ofConferences 4, 00011 (2015)53. Strasdeit, H. ChemBioChem, 6, 801 (2005)
54. Saladino, R., Crestini, C., Cossetti, C., Di
Mauro, E., Deamer, D. Origins of Life andEvolution of Biospheres, 41, 437 (2011)
55. Washington, J. Origins of Life and Evolutionof Biospheres, 30, 53 (2000)
56. Navarro-González, R., Segura, A. In First
Steps in the Origin of Life in the Universe,
Springer: Dordrecht, 201 (2001)
57. Navarro-Gonzalez, R., Basiuk, V. A. In
Exobiology: Matter, Energy, and Informationin the Origin and Evolution of Life in the
Universe, Springer: Dordrecht, 255 (1998)
58. Navarro-González, R., Basiuk, V. A.,
Rosenbaum, M. In Chemical Evolution:
Physics of the Origin and Evolution of Life,
Springer: Dordrecht, 123 (1996)
59. Stetter, K. O. Nature, 300, 258 (1982)
60. Hoshyaripour, G., Hort, M., Langmann, B.,
Delmelle, P. Journal of Volcanology andGeothermal Research, 286, 67 (2014)6