Ignat Ignatov 1, leg V. Mosin 2



1 Scientific Research Center of Medical Biophysics (SRCMB), 1111, Sofia, N. Kopernik, 6, Bulgaria, e-mail:

2 Moscow State university of applied biotechnology, 109316, Moscow, Talalikhina, 33,Russia, e-mail:


Natural prevalence of deuterium 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 = (155,760,05).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 O2O3 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: 2O + D2O = 2DO, causing deuterium at small amounts to be present in water in form of DO, and at high amounts - in form of D2O. The structure of molecules D2O is the same, as that of 2O, with very small distinction in values of lengths of covalent bonds. D2O boils at 101,44 0, freezes at 3,82 0, has density at 20 0 1,105 /sm3, and the maximum of density is not on 4 0, as for usual water, but on 11,2 0 (1,106 /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 2O. Basically, they are reactions catalyzing by ions D3+ or H3+ 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 3O- ion, and D- 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.% D) 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 , 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, halobacteria, blue-green algae) in D2O with using 1H-NNR-, IR-, and mass-spectrometry technique. 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 % D2O) and the subsequent selection of clones resistent to deuterium. Cells grown on media with a low gradient of D2 concentration were transferred on media with big gradient of concentration, up to 98 % D2. 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 D2 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 + 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 23 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 logperiod 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 (HD) 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) 3+ HOH + 2 = Ca(HC3)2

(3) CO2 + - = HC3-

(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(HC3)2 from H2O, 2 and 3. In the presence of hydroxyl OH- ions 2 transforms into HC3- (equation (3). Equation (4) is valid for the process of dolomite formation of stromatolites.

Furthermore, we have carried out the research of mineral, sea and mountain water from Bulgaria by IR-spectroscopy method of differential non-equilibrium energy spectrum (DNES) relative to the control deionized water (fig. 2, curves 1-5, the table). In experiments were investigated samples of water from karst springs. 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% water. The closest to the IR-spectrum of castus juice was the IR-spectrum of the mineral water contacting with 3 (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 (fig. 2, curve 5) was substantially different from the IR-spectrum of sea mineral and mountain water. The values of average energy (∆EH... O) of hydrogen O-bonds between molecules H2O in the process of formation of (H2O)n associates, measured by the DNES method were measured at 0,10670,0011 eV.


Fig. 2. NES 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).

The 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



































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 +, 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 ( 46) these reactions do not occur, whereas alkaline conditions ( 89) promote their course.

In synthesis of organic molecules other energy sources, e. g. geothermal sources could be used. 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 waters boiling point 950 [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, . 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 atmospheres 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.



1. Linsky, J.L. D/H and nearby interstellar cloud structures, Space Science Reviews, NY: Springer Science, Business Media, 2007, V. 130, p. 367; Linsky, J.L. et al. What is the total deuterium abundance in the local Galactic disk? // Astrophysical Journal, 2007, V. 647, p. 1106.

2. Lis G., Wassenaar L.I., Hendry M.J. High-Precision Laser Spectroscopy D/H and 18O/16O Measurements of Microliter Natural Water Samples // Anal. Chem., 2008, V 80 (1), p. 287-293.

3. Mosin O. V. Deuterium, heavy water, evolution and life // Vodoochistka, vodopodgotovka, vodosnabzhenije, 2009. 8, p. 64-70.

4. Lobishev V. N., Kalinichenko L. P. Isotopic effects of D2O in biological systems M.: Nauka, 1978, 215 p.

5. Vertes A. Physiological effects of heavy water. Elements and isotopes: formation, transformation, distribution. - Dordrecht: Kluwer Acad. Publ., 2004, 112 p.

6. Mosin O. V., Skladnev D. A., Shvets V. I. Studying of physiological adaptation to heavy water // Biotechnologija, 1999. 8, p. 16-23.

7. Mosin O. V., Skladnev D. A., Shvets V. I. Methods for production of proteins and amino acids, labelled with stable isotopes 2, 13 15N // Biotechnologija, 1996. 3, p. 12-32.

8. Mosin O. V., Ignatov I. Isotopic effects of deuterium in cells of bacteria and microalgae // Water: chemistry and ecology, 2012. 3, p. 83-94.

9. Ignatov, I., Energy Biomedicine, Origin of Living Matter, Informationability of water, Bioresonance, Biophysical Fields, Institute for Creative Healing, Munich (2007).

10. Ignatov, I., Which water is optimal for the origin (generation) of life? EUROMEDICA, Hanover, (2010).

11. Calvin M. Chemical Evolution, Oxford: Clarendon, 1969, p. 278.

12. Mathews C.N., Moser R. Peptide synthesis from hydrogen-cyanide and water // Nature, 1968, V. 215, p. 1230-1234.

13. Abelson P. Chemical events on the"primitive earth. // Proc. Natl. Acad. Sci. U. S., 1966, V. 55, p. 1365-1372.

14. . Sugawara. Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA, Nature Chemistry, 2011. V. 1127, p. 775-780.