ISSN 0006�3509, Biophysics, 2014, Vol. 59, No. 2, pp. 334–338. © Pleiades Publishing, Inc., 2014.Original Russian Text © S.M. Pershin, E.Sh. Ismailov, Z.G. Suleimanova, Z.N. Abdulmagomedova, D.Z. Zagirova, 2014, published in Biofizika, 2014, Vol. 59, No. 2, pp. 408–413.

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INTRODUCTION

Opening of water channels of ~3 Å diameter inmembranes of the cell, admitting only monomers ofH2O at a rate ~3 · 109 molecules per second [1], hasexpanded the understanding of the biophysics ofexchange processes and elevated the role of watertherein [2, 3]. Special significance at that is possessedby the ability of H2O molecules to create complexesconnected with a hydrogen bond and rearrange theirstructure, and also to reside in free state. Howeverunclear remains [1] the mechanism of destruction ofthe bound state of H2O molecules and liberation ofmonomers of near�cellular water (in complexes ofblood and plasma, and also in hydrate layers of cellmembranes [3]) in the vicinity of water channels forprovision of the indicated rate of H2O transport. Asupposition about rupture of hydrogen bonds for liber�ation of monomers from complexes [1] does not satisfythe law of energy conservation, inasmuch as itdemands the availability of an energy reservoir. Henceit follows also that any treatment of water that leads toan increase of the share of free monomer thereinwould influence the cell metabolism. Therefore newexperimental data pointing to an increase of the rate ofexchange processes of the cell and its productivityupon acting on water by various physical fields wouldtestify to elevation of the share of monomers of H2O inthe vicinity of water channels of membranes. In the

given work account is taken of quantum differences ofspin modification, ortho and para spin�isomers ofH2O.

It is known also that while experimentally studyingthe biophysics of the cell for acceleration or inhibitionof some processes use is made of physiological solutionwith special preparation: bubbling by gases, variationof acidity, content of microelements, salts etc. Forexample, replacement of light isotopes H2O withheavy isotopes D2O in an aqueous solution hasallowed revealing a shift of the temperature of a jumpof deformability of human erythrocytes and their per�meability through microcapillaries from 36.6 to37.2°C [4]. Such replacement during a study of photo�synthesis [5] revealed modulation of the kinetics ofexcitonic state of the reaction center at frequencies ofrotation of H2O and D2O molecules respectively. Ahypothesis was formulated that the spin�isomer orthoH2O executed the role of a mediator [6]. Most signifi�cantly the influence of nuclear spin on processes in thecell was revealed recently by A.L. Buchachenko et al.[7]. They have managed to substantially (two–threetimes) increase the effectiveness of synthesis of ATPsynthase after substitution of 24Mg with 25Mg, having anonzero spin. The physics of this phenomenonremains finally not clear, however experimentally sub�stantiated is the governing role of exactly the nuclearspin of the 25Mg2+ ion in energetic processes in the

DISCUSSIONS

Functional Characteristics of Yeast Cells in Nutrient Aqueous Solution Enriched with Ortho�H2O Isomers

S. M. Pershina, E. Sh. Ismailovb, Z. G. Suleimanovab, Z. N. Abdulmagomedovab, and D. Z. Zagirovab

aWave Research Center, Prokhorov Institute of General Physics, Russian Academy of Sciences, Moscow, 119991 Russiae�mail: pershin@kapella.gpi.ru

bDagestan State Technical University, Makhachkala, Russiae�mail: elderismailov@mail.ru

Received October 12, 2011; in final form, September 18, 2012

Abstract—It has been experimentally established that cultivation of yeast cells in depleted, dietary or normalnutrient aqueous solutions enriched with ortho�H2O spin isomers is accompanied by an increase in theamount of carbon dioxide produced by the cells and an increase in their biomass. It has been revealed that therate of metabolic processes and biological activity depends on the quality of nutrition and enhances in timein both nutrient solutions. In contrast, the reproductive function and the rate of cell division are insusceptibleto the components of nutrition, but intensified in a solution enriched with ortho�H2O similar to retardationof aging. The observed effects are discussed in assumption that an increase of a portion of ortho�H2O mole�cules occurs in the neighborhood of water channels in the cell membrane that let through only monomers ofH2O and determine the rate vicinity of metabolic processes.

Keywords: ortho�H2O isomers, H2O transport via water channels in membrane, increase of biomass andbaker’s yeasts production

DOI: 10.1134/S0006350914020213

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FUNCTION OF YEAST IN MEDIUM ENRICHED WITH ORTHO WATER 335

cell. Possibly, a similar role of mediator is executed byspin�isomer ortho H2O in processes of charge separa�tion in photosynthesis [6].

Earlier [8, 9], by the method of four�photon spec�troscopy we have managed to disclose in water reso�nance lines that were assigned to rotations transitionsof ortho and para spin�isomers of H2O. Recently [10],the availability of H2O monomers in water and ices wasconfirmed in experiments on X�ray beam scatteringspectroscopy. Then [11] we disclosed spin�selectiveinteraction of proteins and DNA with para isomers ofH2O during formation of hydrate shells in aqueoussolutions. This manifested itself as decreasing intensityof the lines of para H2O with simultaneous increase inthe intensity of the lines of ortho isomers of H2O,which pointed to binding of para H2O in the hydrateshell and their conversion into ortho H2O near pro�teins and DNA, which increased the concentration ofortho isomers in biosolution. Apart from that, by non�linear�optical and NMR spectroscopy it was estab�lished [12] that water after cavitational treatment, forexample upon cavitational evaporation, is enriched by12–15% for ortho isomers of H2O in strong electricfields of discharge upon collapse of cavitation bubbles.It is essential that water enriched for ortho isomerspossesses elevated permeability and mobility, whichfollows from the increase of the rate of dissolution ofkidney stones of the type of calcium oxalate [13], andalso allows growing lysozyme crystal of another mor�phology from an aqueous solution enriched for orthoH2O [14].

Let us note that the equilibrium at room tempera�ture ratio of ortho/para isomers 3 : 1 for для H2O in gasphase [15] is violated in liquid phase to the side of par�ticularly nonequilibrium ~1 : 1 [16], which corre�sponds to temperature ~(30–50) K [17]. Hence it fol�lows that a part of molecules in water resides in non�equilibrium, supercooled by –(240–270) K in spintemperature (ortho/para ratio), state [16]. It is naturalto expect that “water” strives to restore the equilib�rium ortho/para ratio at any impact thereon of physi�cal fields (electric, magnetic, electromagnetic: radio,SHF and optical range, acoustic and thermal radia�tion). Possibly, exactly through modification of waterthere was manifestation of the action of microwavesand laser radiation for elevating the viability andincreasing the productivity of yeast and other helpfulmicroorganisms [18], and demonstrated was the per�spective value of their application in biotechnology[19–22].

In the given work the main attention is paid to theinfluence of water enriched for ortho isomers aftercavitational treatment on elevation of biomass andproductivity (amount of released carbon dioxide gas)of yeast cells, as indicators that were studied by us ear�lier [19–22]. The results of preliminary measurementswere discussed at the conference “Molecular Struc�

ture of Water and Its Role in Mechanisms of Bioelec�tromagnetic Phenomena” [23].

EXPERIMENTAL

In the quality of an object of investigation we tookfast�acting yeast “Saf�moment” produced in the townof Uzlovaya, Tula Region. The choice of yeast cells wasstipulated by that earlier we have already worked withthem and possess the method of measurements of theparameters of biological activity [19–22]. It is knownalso [24, 25] that yeast cells come to be a verified testobject in microbiology, on which methods of geneengineering were worked out. Recently [26] namely inyeast cells Saccharomyces cerevisiae a gene was iso�lated responsible for life duration. The authors indetail investigated the aging of yeast cells and its con�nection with the process of reproduction. As a resultthey managed to substantially slow down the processof aging and for the first for a living cell to increase theduration of its life two times. This has served for us anadditional stimulus to use yeast cells for revealing therole of spin�isomers of H2O in cellular exchange andreproductive processes, inasmuch as earlier [9, 11]established was the selectivity of interaction of yeastcells with hydrate shells of biopolymers.

The process of fermentation was actualized by uson nutrient media of two kinds, denoted as:

(a) simple, depleted or dietetic, nutrient medium,including 4 g sugar, 1 g white wheat flour and 1 g dryyeast per 100 mL water;

(b) full�fledged nutrient medium, including 4 gsugar, 1 g white flour, 6 mL molasses, 0.015 mL 20%phosphoric acid and 1 g dry yeast per 100 mL water.

For determination of the character of action of theused water the tests were conducted in the followingtwo variants:

1. Tests with the use of cavitationally treated waterwith pH 5.5–5.7, enriched for ortho isomers of H2O,the procedure of obtaining which is expounded inwork [14]. The gist of it consists in cavitational evapo�ration of distilled water in an ultrasonic fountain withsubsequent condensation. The process is similar todistillation of water, only thermal evaporation isreplaced with ultrasonic evaporation with cavitation[ligis.ru/effects/technics/63/index.htm]. For obtain�ing an aerosol we used a focusing radiator of a concavepiezoceramic plate with a resonance frequency of1.7 MHz. The focal spot of the radiator was disposedsomewhat lower than the surface of water and pro�vided a pressure about 10 MPa in the region of focus.Condensed water was stored in a hermetic polyethyl�ene container (without an air bubble and contact withair) in the course of several days at room temperature.Therewith the degree of enrichment for ortho isomersof H2O as compared with distilled water was preservedat a level of 12–15% [12].

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2. Control tests with distilled or boiled water, whichgave identical, in the limits of measuring accuracy,results.

The observed effect were evaluated by two parame�ters: quantity of released carbon dioxide gas, comingto be an index of productivity and biological activity(process of aging), and biomass of yeast, which comesto be an indicator of reproductive processes. Bothparameters under identical conditions indirectly pointto a change in the rate of exchange processes in yeastcells, and also to an increase or decrease of their bio�logical activity. We have supposed that replacement ofonly water in the nutrient solution will reflect thechange in transport of H2O monomers through waterchannels of the cell membrane and biochemical pro�cesses inside it, and namely: an increase in bothparameters will correspond to elevation of cell pro�ductivity or retardation of aging and stimulation of theprocess of reproduction.

It should be noted that the quantity of forming car�bon dioxide gas characterizes the intensity of fermen�tation and the elaboration of biochemical energy inthe form of ATP [7]. Therewith the rate of formationof carbon dioxide gas reflects the change in biologicalactivity of the cells.

For measurement of the quantity of carbon dioxidegas use was made of a special stand. The inaccuracy ofmeasuring the gas volume was determined, first of all,by instrumental accuracy. In our case it reached0.1 mL in accordance with a specially elaboratedmethod with the use of graduated containers of gascollection, which in detail is expounded earlier in ourwork [20]. In each of the variants we took five tubeseach with stoppers and diversion tubes, into which wepoured 15 mL apiece of yeast thinning. The process offermentation was observed in the course of the first fivehours and upon its completion on the next day in 22 hfrom the beginning of the process. The accuracy of

measuring the quantity of carbon dioxide gasimproved from 0.5 to 0.1% in the course of buildup ofproduct. Therewith the scatter of productiveness of gasby yeast over the aggregate of five tube data did notexceed 10%.

The biomass of yeast was determined with the useof a sufficiently quick and well reproduced volumemethod. The yeast suspension (brew) was poured intospecial measuring glasses for 10 mL and centrifuged at3500 revolutions per minute in the course of 5 min.After centrifugation we measured the height of thesediment with a sharp boundary with the aid of thegraduation scale of the measuring glass itself. Usuallythe flat surface of the sediment was inclined relative tothe axis of the measuring glass (the normal to the sur�face of the sediment did not coincide with the axis).Therefore we measured the minimal and maximalheight of the sediment at opposite sides of the measur�ing glass (sometimes with the aid of vernier calipersproviding a precision of 0.1 mm) and calculated themean value of the height for each measuring glass.Then from these values we calculated the volume ofthe sediment in percent to the whole volume withaveraging over a series of measurements of samples ofone specimen with an error not worse than 0.1%.

RESULTS AND DISCUSSION

The dynamics of the quantity of carbon dioxide gasproduced by yeast on depleted and full�fledged nutri�ent medium is shown in Figs. 1 and 2 respectively,while the change of biomass – in Fig. 3. In all figuresthe numeral 2 denotes the results of measurement ofcontrol samples, while numeral 1 – samples withwater enriched for ortho isomers of H2O.

According to the obtained data, the process of fer�mentation both on depleted, dietetic, and on full�fledged nutrient medium upon usage of water after

50

40

30

20

10

1680 124 20

1

2

Time, h

Volume CO2, mL

Fig. 1. Dynamics of production of carbon dioxide by yeastin solution of dietetic nutrient medium in cavitationallytreated (1) and distilled (2) water.

140

40

80

100

60

1680 124 20

1

2

Time, h

Volume CO2, mL

20

120

Fig. 2. Dynamics of production of carbon dioxide by yeastin solution of full�fledged nutrient medium in cavitation�ally treated (1) and distilled (2) water.

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FUNCTION OF YEAST IN MEDIUM ENRICHED WITH ORTHO WATER 337

cavitational treatment proceeds more actively. Thisdistinction reveals itself in the rate of formation of car�bon dioxide gas as soon as after the first 4 h of fermen�tation on full�fledged nutrient medium (Fig. 2) andincreases with time. Therewith, as it follows fromFigs. 1 and 2, on full�fledged nutrient medium the rateof exchange processes and the integral productivity ofyeast cells in whole is higher three–four times, espe�cially at the initial step, while the sensitivity of cellmetabolism to the type of water vividly revealed itselfby the end of measurements. Thus in 22 h the differ�ence in the quantity of released carbon dioxide gasduring fermentation on full�fledged nutrient mediumwas almost an order higher than on depleted medium.

It is noticeable (see Fig. 3) that production of yeastbiomass is also higher (~11%) upon usage of waterenriched for ortho isomers of H2O, as compared withdistilled water (~9%). Let us note that the increase ofyeast biomass, as distinct from release of carbon diox�ide gas (see Figs. 1 and 2) is less sensitive to the qualityof nutrient medium. On the whole these results areconsistent with data of earlier works about elevation ofthe productivity of yeast cells upon action thereon ofmembranoactive radiations, microwave and laser, andalso biologically active compounds [21, 22].

From Figs. 1 and 2 it is seen also that the process offormation of carbon dioxide gas slows down faster insolutions in distilled water both in depleted and in full�fledged nutrient medium, especially at the last stage ofmeasurements (values of productivity are smaller).Hence it follows that replacement of water with oneenriched for ortho isomers of H2O leads to stable ele�vation of the duration of biological activity of yeastcells, which is similar to retardation of aging [26].

The mechanism of the disclosed phenomenon ofelevation of productivity and duration of biologicalactivity of the cells of baker’s yeast in solutionsenriched for ortho H2O is conditioned, in our opinion,by specific properties of the molecule of ortho H2O,which differ it from the molecule of para H2O [15].Thus a molecule of ortho H2O does not have a zerorotational level of energy [11, 15], therefore the givenmonomer rotates always. The energy of the first rota�tional level of this spin�isomer constitutes 23.8 cm–1

[15–17]. Conversely, a part of molecules of spin�isomerà para H2O does not rotate at room temperatureand resides at zero level in accordance with a Boltz�mann distribution of population over energetic states.Apart from this, spin isomers of H2O have particularlyquantum distinctions: a molecule of ortho H2O pos�sesses a sum magnetic moment (the spins of its protonsare oriented parallel), while the spins of protons ofpara H2O – antiparallel [15, 17]. Therefore moleculesof ortho H2O interact with magnetic and electromag�netic fields, which manifests itself in NMR spectros�copy and tomography [12]. Here it should be under�scored that an increase of the brightness of regions ofan NMR image of an organ obtained in a tomographin units of proton density [12] in fact reflects anincrease of the relative concentration of ortho H2O,

which interact with the magnetic field of the tomo�graph through own sum nuclear magnetic moment[27]. This fact is important for us in that it shows a pos�sibility of prolonged existence of foci with elevatedconcentration of ortho isomers in a living organism.

As mentioned above, it is namely the mobility ofmolecules of ortho H2O that, most likely, provideswater enriched for isomers of ortho H2O by 10–15%with elevated dissolving ability [13] and comes to be aleading factor in the here�considered case. Unclearremains the mechanism of slow relaxation of enrich�ment to initial value. Possibly, a large life time is stipu�lated by that any enrichment of water for ortho iso�mers is energetically more advantageous, inasmuch asit shifts the ortho/para ratio to the side to equilibriumvalue 3 : 1. A key factor for us comes to be that trans�port of water through water channels of molecularmembranes [1], which determine the vital activity ofyeast cells as well, is provided only by monomers ofH2O. Therefore enrichment of water of nutrient solu�tions with monomers of ortho H2O elevates their con�centration also in the vicinity of water channels and,accordingly, permeability of cell membranes.

Possibly, the rate of biochemical reactions withparticipation of ortho isomers of H2O inside the cellalso increases, which influences also the process ofreproduction. When yeast cells start to reproduce, theyactualize a special cycle of cell division – meiosis,which passes through a state of producing spores [26].In Fig. 3 it is seen that yeast biomass, reflecting theprocess of reproduction, practically does not dependon the quality of nutrition. Conversely, replacement ofonly water from distilled to enriched for ortho isomersof H2O accelerates the process of reproduction (pro�duction of spores) and is accompanied by an increaseof biomass. It is quite important to note that accretionof biomass (or increase of the rate of reproductionmore than by 20%) also does not depend on the qualityof nutrition. This fact reflects the fundamental prop�erty of a living cell or organism: the mechanism of

12

10

8

6

4

2

DietNorm

Bio

mas

s, v

ol %

1

2

1

2

11.5% 9.2% 11.3% 9.5%

Fig. 3. Yeast biomass in percent of volume of yeast suspen�sion of dietetic (“Diet”) and full�fledged (“Norm”) nutri�ent medium in cavitationally treated (1) and distilled (2)water in an identical period of time.

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reproduction is the most conservative and stable to thequality of nutrition and external changes of the condi�tions of existence.

No less interesting is a magnetic mechanism ofinfluence of nuclear spin of ortho H2O on the produc�tion of ATP synthase in yeast cells similarly to pro�cesses upon enrichment of solution with ions of mag�nesium with nonzero nuclear spin of 25Mg2+ ion [7].Study of the contribution of this factor in alternatingand permanent magnetic fields will allow evaluating itsmagnitude.

CONCLUSIONS

The selective ability of water channels to letpass only monomers of H2O [1] and enrichment ofcavitationally treated water for ortho H2O monomers[12−14] give ground for supposing that elevation ofbiological activity and processes of reproduction ofyeast cells disclosed by us upon replacement of onlywater in solutions of both depleted (dietetic) and full�fledged nutrient medium is conditioned by elevationof the concentration of ortho H2O in solution. Theseexperimental results have been obtained, as far as weknow, for the first time.

The increase in the rate of reproduction and pro�duction of carbon dioxide gas, pointing to rejuvena�tion of the colony or retardation of aging of cells,demands thorough study (jointly with microbiologicaltests and nanobiological technologies of isolation ofgene NDT80 responsible for processes of aging [26]).

The applied aspect of the obtained results is moreobvious, inasmuch as they may be applied in elabora�tion of new biotechnologies for food and other kinds ofproduction. Doubtlessly, it is important also to knowwhether there is a change or “conservation” of gusta�tory, organoleptic indices in products (bread, kvassand other drinks) prepared on yeast after their modifi�cation in the process of such nano� (scale of yeastcells) and molecular (scale of water molecule) bio�technology, which will come to be a subject of nextworks.

ACKNOWLEDGMENTS

The work was partly supported by the RussianFoundation for Basic Research, grants 09�02�01173,08�02�00008, 10�02�90301�V'et_a.

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