*119632*
ISOTOPIC COMPOSITION OF WATER AND ITS TEMPERATURE IN MODELLING PRIMODIAL
HYDROSPHERE EXPERIMENTS
1 Scientific
Research Center of Medical Biophysics (SRCMB), 1111, Sofia, N. Kopernik, 6,
Bulgaria, e-mail: mbioph@dir.bg
2 Moscow State
university of applied biotechnology, 109316, Moscow, Talalikhina,
33,Russia, e-mail: mosin-oleg@yandex.ru
Natural prevalence
of deuterium (D) makes up approximately 0,015 at. % D, and depends strongly on the uniformity of substance
and the total amount of matter formed in the course of early evolution [1]. Constant
sources of deuterium are explosions of
nova stars and thermonuclear processes occurring inside the
stars. Probably, it could explain a well known fact why the amounts of deuterium are increased slightly
during the global changes of climate in worming conditions. Gravitational
field of the Earth is insufficiently strong for retaining of lighter hydrogen,
and our planet is gradually losing hydrogen as a result of its dissociation
into interplanetary space. Hydrogen evaporates faster than heavy deuterium
which is capable to be collected by the hydrosphere. Therefore, as a result of
this natural process of fractionation of isotopes H/D throughout the process of
Earth evolution there should be an accumulation of deuterium in hydrosphere and
surface waters, while in atmosphere and in water vapor deuterium contents are
lower. Thus, on the planet there is going on a natural process of separation of
H and D isotopes, playing an essential role in maintenance of life on the
planet.
The
absolute contents of deuterium (isotopic shifts, δ, ppm) according to the
international standard VSMOW, corresponding to Pacific ocean water which is
rather stable on isotopic composition, compile D/H = (155,76±0,05).10-6
(155,76 ppm) [2]. For the international standard SLAP of natural water of
Antarctic Region containing less deuterium, the absolute contents of deuterium
compile D/H = 89.10-6 (89 ppm). The average ratio of H/D
in nature compiles 1 : 5700. In natural waters the contents of deuterium are
distributed non-uniformly: from 0,015 at.% D for water from the Antarctic ice –
the most deuterium depleted natural water with deuterium contents in 1,5 times
smaller, than in sea water, up to 0,02–0,03 at.% D for river and sea water.
Thawed snow and glacial waters in mountains and some other regions of the Earth
usually contain on 3–5% less deuterium, than drinking water. On the average, 1
ton of river water contains approximately 150–300 g of deuterium. Other natural
waters contain varying levels of deuterium from δ = +5,0 D, %, SMOW
(Mediterranean Sea) up to δ = -105 D, %, SMOW (Volga River).
The
peposition was made by us that primary water
could contain more deuterium in early stages of evolution, and deuterium was
distributed non-uniformly in hydrosphere and atmosphere [3]. As is known, the
primary reductive atmosphere of the Earth, consisted basically of gas mixture
CO, H2, N2, NH3, CH4, was lacked O2–O3
layer protecting the Earth surface from rigid short-wave solar radiation
carrying huge energy capable to cause photolysis and radiolysis
of water. The processes accompanying accumulation of deuterium in
hydrosphere were solar radiation, volcanic geothermal processes and
electric categories in electric discharges in atmosphere. These natural
processes could lead to enrichment of hydrosphere by
deuterium in the form of HDO which evaporates more slowly then H2O,
and condenses faster. The formation of HDO occurs in D2O–H2O
mixtures via isotopic exchange: Н2O + D2O = 2НDO, causing
deuterium at small amounts to be present in water in form of НDO, and at high
amounts – in form of D2O. The structure of molecules D2O is the same, as that of
Н2O, with very small distinction in values of lengths of
covalent bonds. D2O boils at 101,44 0С,
freezes at 3,82 0С, has density at 20 0С 1,105 г/sm3,
and the maximum of density is not on 4 0С, as for usual water, but
on 11,2 0С (1,106 г/sm3). These effects are
reflected in energy of a chemical bond, kinetics and chemical
reactions rates in D2O–H2O
mixtures. Enzymic reactions in D2O are considerably slowed down.
However, there are also such reactions which rates in D2O are
higher, than in Н2O. Basically, they are reactions catalyzing by
ions D3О+ or H3О+ or OD- and
OH-. According to the theory of chemical bond, breaking up of H–O
bonds can occur faster, than D–O bonds, mobility of ion D3O+ is
lower on 28,5 % than Н3O- ion, and ОD- ion is
lower on 39,8% than OH- ion, the constant of ionization of D2O
is less than constant of ionization of H2O [4]. The maximum kinetic
isotopic effect at ordinary
temperatures in a chemical reaction leading to rupture of bonds involving H and
D was calculated, and the maximum ratio kh/kd in macromolecules is in the range of 6 to 8 for C–H versus C–D, N–H versus N–D,
and O–H versus O–D bonds [5].
Deuterated cells
of various microorganisms adapted to the maximal concentration of D2O
in growth media (95–98 vol.% D2O) are convenient objects for
evolutional and adaptation studies as well as structural-functional studies.
During the cellular growth on D2O media there are synthesized macromolecules
in which hydrogen atoms in carbon skeletons are almost completely replaced on
deuterium. Such deuterated macromolecules undergo the structural-adaptive
modificational changes necessary for normal functioning of cells in the
presence of D2O.
Practical interest
to further applying of deuterated cells of various microorganisms in researches
on their basis mechanisms of cellular adaptation to D2O and
molecular evolution, has predetermined a direction of our studies. The purpose
of the present reseach was studying of isotope effects of deuterium and
conditions of primary hydrosphere (temperature, value рН, isotopic
composition). In frames of the research were studied various samples of water
from Bulgaria.
We have
investigated isotopic effects of deuterium in prokaryotic and eukaryotic cells
of various taxonomic groups of microorganisms realizing methylotrophic,
hemoheterotrophic, photoorganotrophic and photosynthetic ways of assimilation
of carbon substrates (methylotrophic bacteria Brevibacterium methylicum,
chemoheterotrophic bacterium Bacillus subtilis, halobacteria
Halobacterium halobium, blue-green algae Chlorella vulgaris) in D2O
with using 1H-NNR-, IR-, and mass-spectrometry technique. Inorganic
salts and glucose were preliminary cristallized in D2О and dried in vacuum
before using. D2O distilled
over KMnO4 with the subsequent control of isotope enrichment
by 1H-NMR-spectroscopy on device Brucker WM-250 (“Brucker”, Germany)
(working frequency 70 MHz, internal standard Me4Si).
For adaptation to
deuterium were used solid 2% agarose growth media with gradually increasing
concentrations of D2O, combined with the subsequent selection of
clones resistent to deuterium. The method of step by step adaptation is
developed for adaptation of cells of various microorganisms to D2O
consisting in plating initial cells on firm (2% agarose) growth media with
increasing gradient of D2O concentration (from 0; 24,5; 49,0; 73,5
to 98 vol.% D2O) and the subsequent selection of clones resistent to
deuterium. Cells grown on media with a low gradient of D2О concentration
were transferred on media with big gradient of concentration, up to 98 vol.% D2О. Degree of cell
survive on maximum deuterated media was about 40%.
Our experiments
demonstrated, that the effects observed at the cellular growth on D2O
possess complex multifactorial character connected to changes of morphological,
cytologic and physiological parameters – magnitude of the log-period,
time of cellular generation, outputs of biomass, a ratio of amino acids, protein,
carbohydrates and lipids synthesized in D2O, and with an
evolutionary level of organization of investigated object as well. The general
feature of bacterial growth in D2О was the proportional increase in
duration of the log-period and time of cellular generation at reduction of
outpunts of a microbic biomass. The experimental data testify that cells
realize the special adaptive mechanisms promoting functional reorganization of
work of the vital systems in the presence of D2O. Thus, the most
sensitive to replacement of Н+ on D+ are the apparatus of
biosynthesis of macromolecules and a respiratory chain, i.e., those cellular
systems using high mobility of protons and high speed of breaking up of
hydrogen bonds. Last fact allows consider adaptation to D2O as
adaptation to the nonspecific factor effecting simultaneously functional
condition of several numbers of cellular systems: metabolism, ways of
assimilation of carbon substrates, biosynthetic processes, and transport
function, structure and functions of macromolecules. There is evidence that
during adaptation to D2O the ration of synthesized metabolites is
changing. Furthermore, deuterium
induces physiological,
morphological and cytological alterations
in the cell. This leads to the formation
in D2O of large atypical cells
[6, 7]. They are usually 2–3 times larger in size and have a
thicker cellular wall compared to the control cells grown on H2O. The structure of DNA in deuterated cells in D2O may alters; distribution
of DNA in them was non-uniform. The data obtained confirm that
adaptation to D2O is a phenotypical
phenomenon as the adapted cells return back to normal growth after some
log–period after their replacement into H2O. At the same time the
effect of convertibility of growth on H2O/D2O does not
exclude an opportunity that a certain genotype determines displaying of the
same phenotypical attribute in D2O.
Experiments with D2O
have shown (fig. 1), that green-blue algae is capable to grow on 70% D2O,
methylotrophic bacteria – 75% D2O, chemoheterotrophic bacteria – 82%
D2O, and photoorganoheterotrophic bacteria – 95% D2O.
Fig. 1.
Survival rate of cells of the studied microorganisms in water with various
content of deuterium.
In the process of
adaptation to D2O the most important for macromolecular structure
are dynamic ahort-lived hydrogen (deuterium) bonds formed between the neighbor
atoms of H(D) and O, C, N, S- heteroatoms, playing an essential role in
maintenance of spatial structure of macromolecules and intermolecular
interactions. The substitution of H
with D atom affects the stability and geometry of hydrogen bonds in apparently
rather complex way and may, through the changes in the hydrogen bond zero-point
vibrational energies, alter the conformational dynamics of hydrogen
(deuterium)-bonded structures of DNA and protein in D2O. It may
cause disturbances in the DNA-synthesis, leading to permanent changes in DNA
structure and consequently in cell genotype. The multiplication which would
occur in macromolecules of even a small difference between a proton and a
deuteron bond would certainly have the effect upon the structure. The
sensitivity of enzyme function to the structure and the sensitivity of nucleic
acid function (genetic and mitotic) would lead to a noticeable effect on the
metabolic pathways and reproductive behavior of an organism in the presence of
D2O. And next, the changes in dissociation constants of DNA and
protein ionizable groups when transferring the macromolecule from H2O
to D2O may perturb the charge state of the DNA and protein
molecules. Other important property is defined by the three-dimensional structure of D2O
molecule having the tendency to pull together hydrophobic groups of macromolecules to minimize their disruptive
effect on the hydrogen (deuterium)-bonded network in D2O. This leads to stabilization of the
structure of protein and nucleic acid macromolecules in the presence of D2O
[8]. At placing a cell in D2O, not only H2O is removed
from a cell due to reaction of D2O dissociation, but also there is
occurred fast isotopic (H–D) exchange in hydroxyl (-OH), sulfhydryl (-SH)
and amino groups (-NH2) of all organic substances, including
proteins, nucleic acids, carbohydrates and lipids. It is known, that in these
conditions only covalent C–H bond is not exposed to isotopic (H–D) exchange
and, thereof only substances with bonds such as C–D can be synthesized de
novo [9].
Biological
experiments with D2O
and structural-conformational studies enable to modelling conditions under
which life has evolved. The most favorable are accepted alkaline mineral waters
interacting with CaCO3 and then sea waters [10]. Circulating in
bowels on cracks, crevices, channels and caves karst waters are enriched with
Ca(HCO3)2, actively cooperating with live matter. Once
appeared in these waters the process of self-organization of primary organic
forms in water solutions may be supported by thermal energy of magma, volcanic
activity and solar radiation.
In
connection with these data are important the following reactions:
(1) CO2
+ 4H2S + O2 = CH2O + 4S + 3H2O
(2) СаСО3+ HOH + СО2 = Ca(HCО3)2
(3) CO2 + ОН- = HCО3-
(4) 2 HCO3- +
Ca2+ = CaCO3 + CO2 + H2O
The equation (1) shows how some chemosynthetic bacteria use energy from
the oxidation of H2S to S. The equation (2) is related to formation
of Ca(HCО3)2
from H2O, СО2 and СаСО3. In the presence
of hydroxyl OH- ions СО2 transforms into HCО3- (equation
(3). Equation (4) is valid for the process of dolomite formation of
stromatolites.
Furthermore, we
have carried out the research of mineral (fig. 2, curve 2), sea water
(fig. 2, curve 3) and mountain water (fig. 2, curve 4) from
Bulgaria by IR-spectroscopy method of differential non-equilibrium energy
spectrum (DNES), which was performed on Fourier-IR spectrometer Brucker Vertex (“Brucker”, Germany)
(a spectral range: average IR – 370–7800 sm-1;
visible – 2500–8000 sm-1; the permission – 0,5 sm-1;
accuracy of wave number – 0,1 sm-1 on 2000 sm-1). As a
control it was used deionized water (fig. 2, curve 5). Also IR-spectra
of castus juice were investigated by DNES method (fig. 2, curve 1). The
cactus was selected as a model system because the plant contains about 90% H2O.
The closest to the IR-spectrum of castus juice was the IR-spectrum of the
mineral water contacting with СаСО3 (fig. 2, curve
2). IR-spectra of plant juice, mineral water and water of the kars springs
have magnitudes of peaks in IR-spectra
at -0,1112; -0,1187; -0,1262; -0,1287 and -0,1387 eV, accordingly. Similar
peaks in the IR-spectrum between cactus juice, mountain and sea water were
detected at -0,1362 eV. The IR-spectrum of the control sample of deionized
water was substantially different from the IR-spectrum of sea mineral and
mountain water (Table). The values of average energy (∆EH... O)
of hydrogen Н…O-bonds
between molecules H2O in the process of formation of (H2O)n
associates, measured by the DNES method were measured at 0,1067±0,0011
eV.
Fig. 2. ДNES spectra of water of various origin: 1 – cactus juice; 2 – mineral water Rupite (Bulgaria); 3 – sea water (Varna, Bulgaria); 4 – mountain water (Teteven, Bulgaria); 5 – deionized water (control).
Table. Characteristics of IR-spectra of
water of various origin obtained by DNES-method.
-Ex (eV) Cactus juice |
-E (eV) Mineral
water Rupite |
-E (eV) Sea water
|
µm |
cm-1 |
0,1112 |
0,1112 |
|
11,15 |
897 |
0,1187 |
0,1187 |
|
10,45 |
957 |
0,1262 |
0,1262 |
|
9,83 |
1017 |
0,1287 |
0,1287 |
|
9,64 |
1037 |
0.1362
|
|
0,1362 |
9,10 |
1099 |
0,1387 |
0,1387 |
|
8,95 |
1117 |
The
data obtained proved that hot mineral alkaline water is preferable for
maintanence of life. These data also can predict a possible way of transition
from synthesis of small organic molecules due to the energy of UV solar
radiation and thermal activity to more complex organic molecules as protein and
nucleic acids. The important factor in reaction of condensation of two
molecules of amino acids is allocation of H2O molecule when peptide
chain is formed. As reaction of polycondensation of amino acids is accompanied
by dehydratation, the H2O removal from reactional mixture speeds up
the reaction rates. This testifies that formation of organic forms may occur
nearby active volcanoes, because at early periods of geological history
volcanic activity occurred more actively than during subsequent geological
times. However, dehydratation accompanies
not only amino acid polymerization, but also association of other blocks into
larger organic molecules, and also polymerization of nucleotides into nucleic
acids. Such association is connected with the reaction of condensation, at
which from one block removes proton Н+, and from another
– hydroxyl group (OH-) with formation of H2O molecule.
The
possibility of existence of condensation-dehydratation reactions under conditions of primary hydrosphere was
proven by Calvin in 1965 [11]. From most chemical substances hydrocyanic acid
(HCN) and its derivatives – cyanoamid (HNCN2) and dicyanoamid
(HN(CN)2) possess dehydratation ability and the ability to catalyze the
process of linkage of H2O from primary hydrosphere [12]. The
presence of HCN in primary hydrosphere was proven by Miller's early
experiments. Chemical reactions with HCN and its derivatives are complex with
chemical point of view; in the presence of HCN, HNCN2 and HN(CN)2 the condensation of separate blocks
of amino acids accompanied by dehydratation, can proceed at normal temperatures
in strongly diluted H2O-solutions. Furthermore, polycondensation of
amino acids in the presence of HCN and its derivatives depends on acidity of
water solutions in which they proceed [13]. In acid water solutions (рН 4–6) these reactions do not occur, whereas
alkaline conditions (рН 8–9)
promote their course.
In
synthesis of organic molecules other energy sources, e. g. geothermal sources
could be used. Thus, in solutions of formaldehyde CH2O with hydroxylamine NH2OH,
formaldehyde with hydrazine (N2H4) in water solutions with НCN, after heating
of a reactionary mixture to 950С amino acids were detected and even
polimerized into short peptide chains that is the important stage towards
inorganic synthesis of protein [14]. In a reactionary mixture with solution HCN
in water NH3 were formed purines and pyrimidines [15]. In
other experiments amino acid mixtures were subjected to influence of
temperatures from 600C up to
170 0С with
formation of short protein-like molecules resembling early evolutionary forms
of proteins subsequently designated as thermal proteinoids [16]. Under cetrain
conditions in hot mixture of proteinoids in water solutions are formed
elementary membrane like
proteinoid microspheres with diameter 5–10 µm [17]. In 2011 a team
of Japanese scientists led by T. Sugawara created a membrane like proto cells
from aqueous solution of organic molecules, DNA and synthetic enzymes
under temperature close to water’s
boiling point 950С [14]. These laboratory experiments is an excellent
confirmation of the possibility that life originated in hot water.
The data obtained testify that life maintanence depends on phisical-chemical properties of water and external factors – temperature, рН. Hot
mineral alcaline water, which interacts with CaCO3 is closest to
these conditions. Next in line with regard to quality is sea and mountain
water. In
warm and hot mineral waters IR-peaks in DNES spectra
were more
expressed in comparison with the IR-peaks received in the
same water with lower temperature. The
spectral range of DNES was in the middle infrared range from 8 to 14 mm. It is thought that there is the Earth
atmosphere’s window of transparency for the electromagnetic radiation in the
close and middle infrared range. In this interval energy is radiated from the
Sun towards the Earth, and from the Earth towards surrounding space. If in the
primodial hydrosphere was much more deuterium, this is a significant fact
regarding thermal stability of deuterated macromolecules in the preservation of
life under thermal conditions.
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