*For offprint requests: Department of Botany, University of Toronto, Toronto, ON, Canada M5S 3B2; fax: (416)978-5878; email: kushner@botany.utoronto.ca

SUMMARY

Some of the environmental, medical, biochemical and other commercial uses of deuterium (D) and heavy water (D₂O) are briefly reviewed. Many microorganisms can grow in "pure" (99.6-99.8%) D₂O, usually after a period of adaptation. Such organisms can produce a very large number of deuterated compounds.

Deuterated glycerol has interesting potential uses as a precursor of transparent plastics and other compounds. We report experiments on the formation of deuterated glycerol by Dunaliella spp, salt-tolerant algae growing in D₂O. These algae produce glycerol as a "compatible solute" when growing in high NaCl concentrations. Although the different species studied did not grow as well in D₂O as in H₂0, the total glycerol production of at least one species was just as high. Our results indicate a new and interesting biotechnological potential for such algae.

Key words: deuterium, heavy water, D₂O, glycerol, algae, Dunaliella.

INTRODUCTION

Deuterium (²H or D), the isotope of hydrogen containing two neutrons and one proton, was separated by Urey et al. (1932). D₂O makes up a small but significant fraction (ca. 0.015 mol %) of natural water. Its ability to act as a moderator of nuclear reactions led to major industrial efforts to produce it in large quantities. It may be concentrated from natural water by several different methods. These include exchange between deuterated hydrogen (HD), hydrogen sulfide (in the Girdler-Sulfide process, which has been most used), ammonia or methylamine and liquid water or water vapor, coupled with the use of continual enrichment processes to make the end product, 99.8% D₂O (Benedict et al., 1981; Miller and van Alstyne, 1994; Rae, 1991). By 1991, sixty years after its discovery, about 29,000 tonnes of D in the form of D₂O had been produced, 60% of this in Canada (Rae, 1991).

All methods are carried out on a very large industrial scale, involving large energy utilization. The increase of the cost of D₂O from $60 US/kg in 1960 to about $240 US/kg in 1980 was mainly due to the increased costs of energy (Rae, 1991).

Since this conference is largely concerned with the risks of releasing different substances into the environment, it should be stated at the beginning that D₂O itself poses little such risk--so long as it has not been used in nuclear reactors. As will be seen below, D₂O itself is not toxic to animals, except under

exceptional and very improbable circumstances. It is non-radioactive. The amount of the radioactive isotope, 3H or tritium, found in heavy water, is very low, < 6kBq/kg D₂O (< 0.15 µCi/kg D₂O), barely above background. However, in nuclear reactors, neutron bombardment may convert D to 3H, so that levels of 0.6 TBq/kg (15 Ci/kg) or more can be reached, an increase of 8 orders of magnitude (Homma and Murase, 1990). This review will not be concerned with hazards of leaks of ³H-containing heavy water or other dangerous substances from nuclear reactors.

As large amounts of D₂O became available, very many lines of research were pursued on deuterated compounds, and many uses have been found for such compounds.

Space limitations make it impossible to give comprehensive references for all the points listed. We hope to prepare a bibliography on uses of D, D₂O and deuterated compounds to be sent to those requesting it.

Protective and therapeutic uses. Partly because of its intrinsic structure (Katz, 1965), D₂O increases the stability of organic molecules, macromolecules, viruses and vaccines. This can have important practical effects when vaccines and other heat-sensitive therapeutic substances must be distributed in developing countries, many of whose districts may lack electricity and refrigeration. A number of patents exist for biostabilizing ability of heavy water. These describe increased thermal and microbial stability and slower disaggregation of certain macromolecules (e.g., Crainic and Simpson, 1994). The presence of 95% D₂O was found equivalent to a 4-5C reduction in storage temperature relative to H₂O, for macromolecular pharmaceuticals such as vaccines; as little as 7-25% D₂O helps prevent protein denaturation. D₂O medium has been recommended for storing organs, tissues, tissue parts, proteins or enzymes, sera and blood preparations and for the perfusion of organs intended for transplants (Wenzel, 1974), microorganisms and enzymes (Hamaya and Horikoshi, 1989), and hydrolysable compositions such as alpha-hydroxy-carboxylic acids (especially benactyzine) (Teva Pharm. Ind. Ltd. 1989).

Heavy water is also cryoprotective and may shield erythrocytes and other cells, cell constituents, organ-parts or organs against damage by low temperature (<-26C) storage (Boehringer Sohn C H GMBH, 1976).

Though D₂O stabilizes proteins and other macromolecules, it does not always make living cells more heat-stable, and may make them less so (reviewed by Unno et al., 1989). The latter effect may be partly due to the lowered ability of deuterated cells to synthesize heat shock proteins and other chaperoning (Unno and Okada, 1991; 1994; Yokogaki et al., 1995). Some direct therapeutic uses of heavy water have been explored, in both animals and humans. Non-toxic levels of heavy water reduce induced hypertension and associated changes in rats (Vasdev et al., 1993; 1994). A patent has been obtained for the use of heavy water in treating human subjects (Liepins, 1993).

Boron neutron capture therapy (BNCT) is used to treat tumors, especially brain tumors, by neutron irradiation of boron-containing compounds bound to malignant cells. Subsequent emission of -particle and other radioactive rays selectively kills these cells. The degree of neutron penetration into tissue, which limits this method, can be greatly increased if the patient's brain is "loaded" with heavy water (Hatanaka, 1989, 1991).

Heavy water has been widely used in measurements of body water space, in children and in adults at various stages of nutrition and exercise. Many human subjects have been injected with or have swallowed a few ml (often 0.1 ml per kg body weight) of heavy water. This increases the D₂O content in the blood from 150 to about 300 ppm, which subsequently decays to the normal level with a half-life of a few days (Coward, 1979). The water space of animal bodies may be determined by the degree of dilution of the heavy water, which can be measured with great sensitivity.

The widespread use of heavy water in clinical studies and treatments without reported adverse effects implies that it is not very toxic to humans. This is also suggested by animal experiments, in which toxic effects did not begin to appear until the D content of blood and body fluids and tissues was over 20% (Katz, 1960; 1965; Thomson, 1963). To attain such a concentration and adult human weighing about 70 kg would have to drink rapidly more than 10 L D₂O.

Microbial growth in D₂O. D₂0 is even less toxic to microorganisms than to multicellular creatures. Some bacteria and algae can grow in "pure" (normally 99.6-99.8%) D₂O, though usually more slowly than in H₂O, and after a period of adaptation. Even more complex cells, such as protozoa can grow in quite high concentrations (70-100% D₂O) (Katz, 1965; Thomson, 1963). Microorganisms that can grow in pure D₂O include, but are not limited to, the algae, Chlorella and Scenedesmus (Katz, 1965; Unno et al., 1987, 1989) and, as this report shows, the salt-tolerant Dunaliella species. Bacteria include Escherichia coli, Pseudomonas putrefaciens, Serratia marcescens (Katz, 1965; Thomson, 1963; Vanatulu et al., 1993), and extremely halophilic archaebacteria (Crespi, 1982). Among yeasts and fungi, Torula and Aspergillis spp. have been grown in pure D₂O (Thomson, 1963), as have methylotrophic Candida spp. (Haon et al., 1993). In most cases, a period of "training" or adaptation is required at lower D₂O concentrations before cells can grow in the pure substance; mechanisms involved in such adaptation have hardly been studied. Microorganisms growing in D₂O can provide a large number of deuterated compounds. Algae that have grown in D₂O can provide biomass to serve as feedstock for the growth of heterotrophic microorganisms (Crespi, 1988).

Environmental and pollutant studies. Determination of the natural occurrence of deuterium and other stable isotopes is a powerful means for investigating past and current environmental changes in the Earth's atmosphere and hydrosphere. The isotopic composition of trace atmospheric constituents provides information on sources, sinks and transformations of these compounds and, since the distribution of environmental isotopes is governed by environmental conditions, they can be used to examine both natural and anthropogenic influences on climatic variation (Kaye, 1992; Rozanski and Gonfiantini, 1990; Bowen et al., 1990). Stable isotopes of H, O and C have been used to assess the atmospheric methane inventory (Levin and Doerr (1991), to evaluate the source of water vapour in the upper troposphere (Smith, 1992), and by studies of the deuterium profile of an Antarctic ice core, to assess atmospheric temperature changes over 160,000 years (Jouzel et al., 1987).

Other such studies have provided valuable information about the steady-state characteristics and past changes in the water cycle, including information on ground- and soil-water sources and movement (Bowen et al., 1990; Wood and Sanford, 1995; Sadler et al., 1992; Jacob et al., 1992), lake hydrology (Krabbenhoft et al., 1991; Aly et al., 1993) and evaporation rates in deserts (Sonntag et al., 1984; Nativ et al., 1995).

D₂O and D₂¹⁸O (double-labelled water) have been used to study snowmelt contributions to spring runoff in acid-stressed headwater streams (Wels et al., 1988), in differentiating different source waters from background groundwater and for determining probable contaminant sources associated with a landfill operation (Mohr et al., 1992), and for estimating native interstitial water and moisture migration in petroleum deposits (Fjerstad et al., 1993; Karasev and Dubinchuk, 1977).

Evapotranspiration, whose magnitude is influenced by growing-season precipitation, humidity and temperature, enriches D and ¹⁸O in leaf water of plants (Cooper and DeNiro, 1989; Ramesh et al., 1985). D/H ratios of tree sap can be used to determine the relative contribution of summertime rains and groundwater as the tree's source water (White et al., 1985). Isotopic (²H and ¹⁸O ratios in plant leaves can be used to determine sources of water, water movement and changes of evaporation patterns during the day (Bariac et al., 1994a and b).

D₂O has also been used to follow water behaviour in materials. Water distribution in concrete samples has been spatially resolved using magnetic resonance imaging, with D₂O employed as a tracer (Kaufmann et al., 1992).

Stable isotopes have also been applied to the investigation of mineral resources (Toulhoat et al., 1990; Wilson, 1989; Brandt et al., 1986). D, ¹³C and ¹⁸O have been used to establish the relative contributions of thermogenic versus biogenic processes to the formation of natural gases in petroleum deposits (Feinstein et al., 1993; Ahmad and Alam, 1992). After organic matter is formed, environmental changes have minimal effect on ¹³C composition, but can greatly modify deuterium content (Missbach and Schatze, 1987).

Deuterated molecules make useful, very sensitively-detected tracers for environmental pollution. To take only one example among many, Thierrin et al. (1995) used deuterated benzene, toluene, p-xylene and naphthalene to study movement and degradation of these compounds in a plume of contaminated groundwater.

Sources of foods and organic chemicals. The D/H ratios of hydrocarbons can indicate the origin of wines and spirits, petroleum products, rubber, as well as sugar or ethanol in foods and beverages (Martin et al., 1986; Martin and Martin, 1983, 1991). Remaud et al., (1992) used information on site-specific D content and overall ¹³C content of acetic acids to differentiate between natural and synthetic (fossil origin) vinegars, or mixtures of the two.

H₂O leaves plants more readily than D₂O, and hence plant juices will be richer in D₂O than water entering the plant. This can be used to distinguish fruit juices reconstituted from concentrates from the juices found in Nature (Roth, 1993).

Biochemical and structural studies. A vast amount of chemical and biochemical work has taken advantage of the fact that D from D₂O can readily exchange with H atoms of organic molecules, especially those bound to O and N atoms. The exchangeability can show when certain groups in proteins and antigen/antibody complexes, are exposed to water (Englander and Mayne, 1992: Paterson et al., 1990). Since C-D bonds are much stronger than C-H bonds, relative reactivity of deuterated compounds can help determine to what extent C-H bond breakage is normally involved. Other aspects of these "deuterium isotope effects" have been discussed in great detail (Cleland et al., 1976; Gillette et al., 1994; Klein et al., 1995; Vanoni et al., 1990). Deuterated compounds are extensively used in spectrophotometric studies. Since H₂O interferes with nuclear magnetic resonance (NMR) signals, this powerful technique for studying molecular structure is often applied to compounds suspended in D₂O or deuterated organic solvents (Hore, 1989; Markley, 1989; LeMaster, 1990.) Deuterated proteins and other macromolecules may be especially valuable for NMR and neutron diffraction studies (Rösch, 1986; Vanatulu et al., 1993).

Substances produced by deu-terated microorganisms. Kyle et al. (1988) described growth of algal species on 99% D₂O. A Neochloris sp. was able to grow well and produced more lipid than during growth in H₂O. Such algae were of special interest as sources of deuterated lubricants. These were shown by previous workers to be many-fold more resistant to thermooxidative breakdown than hydrogenated lubricants. Preparation of deuterated lipids by treating the unsaturated forms with D₂ gas is a much more expensive process than the biological process described. Nevertheless, a market has still not developed for deuterated lipids, largely because their cost is still high. Haon et al. (1993) showed that methyltrophic yeasts growing in D₂O were good sources for deuterated ergosterol.

Deuterated drugs. A great number of deuterated drugs have been synthesized, in order to study their movement and metabolism in bodies of humans and other animals. Of special interest are drugs whose therapeutic properties are changed by deuteration. These include amphetamines, which are more readily transported into the brain in the deuterated form (Wenzel, 1989); halogenated anaesthetics, such as selvoflurane, which, when deuterated are no longer oxidized to toxic forms within the body (Baker et al., 1993), and certain antimicrobial compounds [long-chain fatty acids (Abrahamson et al., 1982) and D-fluorophenylalanine (Merck and Co., 1977)] whose deuterated forms are not oxidized by the affected microorganism arid hence are toxic for longer times

Other uses for D and deuterated compounds. Synthetic deuterated fibres may be incorporated in currency notes. This provides a method of tagging large amounts of currency (for example from drug smuggling) whose transfer is of dubious legality (Muller 1991).

Very recently it was found that using D₂ instead of H₂ to coat metal oxide semi-conductor transistors (such as computer chips) extends their lifetimes many-fold (Lyding et al., 1996) though possibly not enough to account for the changes in technology and fashion that make the most modern computers so soon obsolete.

Deuterated compounds for optical technology. In recent years patents have been obtained for the use of deuterated polymers and other compounds in laser and optical technology. These include deuterated 4-'nitrobenzylidene-3-halo -alkanoylamino-4-methoxy aniline crystals for nonlinear optical devices used for optical communication or information processing and optical recording material (Toray Ind. Inc. 1995), deuterated N, N'-diamino dicarboxylic acid amides with similar uses (Mitsui Petrochem. Ind. Co. Ltd., 1994), deuterated polyvinyl alcohols or polymethaerylic acids for optical recording devices (Iwamoto et al., 1992) and deuterated 1-arginine phosphate frequencyconversion crystals for use in lasers (Barker et al., 1992). Deuterated acrylate polymers have been claimed to be specially useful in optical fibres (Kitahara et al., 1984; Mitsubishi Rayon KK, 1984).

We have been espeially interested in the use of microorganisms as sources of deuterated compounds. Our experiments have involved the formation of a potentially useful product, deuterated glycerol, by salt-tolerant algae growing in heavy water.

The algae selected for this study were different species of the genus Dunaliella, including Dunaliella tertiolecta, D. salina and D. bardawil. These are unicellular eukaryotic green algae, motile by a pair of anterior flagella. Dunaliella species are found in the sea and many salt lakes, such as the Dead Sea in Israel, the Great Salt Lake in Utah, USA, and in many salterns where salt is prepared by evaporating sea water. They have the characteristic, in common with other salt-tolerant algae and yeasts, of producing high concentrations of glycerol as a compatible solute, that is a solute which maintains the internal osmotic pressure equivalent to that outside the cell and also permits the cell's internal enzymatic and other processes to function. ln high salinity, the internal glycerol can make up to 85% of the dry weight of the cell (Brown, 1990; Javor, 1989). If these algae can grow in pure D₂O (with CO₂ as a carbon source), their glycerol (and other molecules) should exist in a deuterated form.

Biological production of deuterated glycerol was of special interest because this substance can be converted to deuterated acrylic acid by the following steps, catalyzed by bacterial enzymes:

Glycerol Ô Propane di-ol Ô 3-hydroxypropionaldehyde Ô Acrylic acid (CH₂ =CH-COOH) (Vancauwenberge et al., 1990).

MATERIALS AND METHODS

Species studied. Dunaliella bardawil was originally obtained from the American Type Culture Collection (ATCC 30861). D. tertiolecta Butcher (UTEX LB 000) was obtained from Dr. J. Hellebust, Department of Botany, University of Toronto. D. salina (UTEX LB 635) was obtained from the University of Toronto Culture Collection (UTCC). Some studies were also carried out on another salt-tolerant alga, Stephanoptera sp. (UTEX LB 635) obtained from UTCC. However, this grew very poorly in D₂O and produced little glycerol, so that results with it are not given.

Cultures were grown in 50 mL liquid Dunaliella culture medium of Ben-Amotz et al. (1989), with NaCl concentrations of 0.5 - 3.5 M, as specified, in 150 mL cotton-plugged Erlenmeyer flasks; the inoculum was, at first, 2 mL of a culture in H₂O and 10 mL of a culture in D₂O (because of lower growth in the latter); as the cultures became adapted to D₂O smaller inocular were used. Cultures were maintained in a growth chamber at 26C on a 14 hr light/ 10 hr dark schedule. Irradiance was by combined cool white fluorescent light and incandescent light at ¹⁸0 µEm^-2s^-1.

Glycerol was usually assayed as a diol after oxidation with periodate (Ben-Amotz and Avron, 1978). In a few experiments, a more specific method involving coupled glycerokinase and glycerophosphate dehydrogenase (Wieland, 1983) was used. Both gave the same results, showing that the di-ol produced was indeed glycerol.

RESULTS AND DISCUSSION

Adaptation of Dunalielia spp. to heavy water. None of the species tested could grow in 100% (actually 99.6%) D₂O when first tested. However, after a process of adaptation involving culturing in higher and higher D₂O concentrations, all the Dunaliella spp were able to grow in 100% D₂O. This process is shown in Figure 1 for D. tertiolecta, and a similar process occurred with the other two species. As the figures shows, with this alga, good growth occurred in 25% and 50% D₂O, on inoculation from H₂O. This growth was at the same level as in H₂O (not shown). No growth occurred when cells were transferred from H₂O to 75% or 85% D₂O (not shown), but cells cultured in 50% D₂O could grow in these higher concentrations. Preculture in yet higher D₂O concentrations led to the ability to grow in still higher concentrations; thus 85% D₂O inoculated from 85% D₂O permitted better growth than the same concentration inoculated from 50% D₂O. Though growth finally occurred in 100% D₂O, this was considerably slower than in lower D₂O concentrations or in H₂O. This was also true of the other species, after adaptation (Figure 2).

As Figure 3 shows, the relative response to different NaCl concentrations of D. bardawil was similar in H₂O and D₂O, and similar results were found for the other Dunaliella spp (not shown).

Increasing two of the essential nutrients, SO₄^-- and NO₃^-, in amounts that increased the level of these substances in the medium five-fold, had no significant effect on growth of D. bardawil in D₂O (not shown).

Effects of external NaCl concentration on glycerol production. This was studied in all three Dunaliella spp; results are summarized in Tables 1 and 2. D. bardawil was studied in more detail than the other species. A somewhat different relation between NaCl concentration and intracellular glycerol production was observed in the two kinds of water. In H₂O there was an almost linear relationship between medium salt concentration and internal glycerol, with most glycerol being found in the cells at the higher salt concentration. In D₂O, such a relationship was not observed, but most internal glycerol was produced at the salt concentrations where the cells were at the most stress (i.e., at the lowest and highest NaCl concentrations permitting growth.) However, total glycerol production also followed the external NaCl concentration. In general, more external glycerol was found in cultures growing in D₂O than in H₂O. Though growth was considerably less in D₂O than H₂O, total glycerol production was approximately equivalent in both cultures of D. bardawil.

Less total glycerol was produced in D₂O than H₂O by D. tertiolecta growing in 0.5 M NaCl and D. salina growing in 2.0 M NaCl. However, the amount of total glycerol produced per cell by both algae was clearly greater in D₂O than in H₂O.

Our experiments have shown that algae which can grow in the relatively extreme conditions of high NaCl concentration are still able to do so in 100% D₂O. They also show that during this process, the algae produce substantial amounts of deuterated glycerol, a potentially valuable product. Indeed, in terms of cell growth, the algae produce more than they would in H₂O. These results add further support to the idea that there are interesting biotechnological potentials in such microorganisms.

ACKNOWLEDGMENTS

This work was partly supported by a grant from the Natural Sciences and Engineering Research Council of Canada to D.J.K.

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Table 1. Glycerol production in H₂O

Test organism	NaCl conc. (M)	Extracellular glycerol (µg/ml medium)	Intracellular glycerol (µg/cell)	Cell density (x106 cells/ml)	Total glycerol (µg/ml culture)
Dunaliella salina	0.5	54.8	28.8	0.65	73.4
(16 days growth)	1.0	205.4	52.2	1.27	271.6
	1.5	305.7	115.6	1.54	483.7
	2.0	274.0	124.4	1.40	448.1
	2.5	212.4	129.1	1.12	357.0
	3.0	169.0	159.1	0.95	320.1
	3.5	121.8	266.0	0.36	217.5
Dunaliella bardawil	0.5	29.4	33.2	0.79	55.6
(17 days growth)	1.0	94.5	69.5	1.21	178.5
	1.5	123.9	65.8	1.24	205.7
	2.0	138.9	79.0	1.27	239.0
	2.5	141.4	80.8	1.41	255.1
	3.0	99.7	108.4	1.12	220.8
	3.5	69.9	129.0	0.9	189.8
Dunaliella tertiolecta	0.5	141.3	12.8	3.35	184.2
(17 days growth)	1.0	206.9	19.7	2.31	252.4
	1.5	150.8	18.2	1.35	175.5
	2.0	202.8	39.7	1.12	247.4
	2.5	227.7	45.9	1.30	287.6
	3.0	93.4	73.1	0.55	268.2
	3.5	no growth	--	--	--

Table 2. Glycerol production in D₂O.

Test species	NaCl conc. (M)	Extracellular glycerol (µg/ml medium)	Intracellular glycerol (pg/cell)	Cell density (x106 cells/ml)	Total glycerol (µg/ml culture)
Dunaliella bardawil	0.5	86.3	565.3	0.008	90.5
(24 days growth)	1.0	105.9	282.4	0.050	120.0
	1.5	121.3	469.9	0.052	145.8
	2.0	120.6	186.0	0.145	147.5
	2.5	131.9	210.5	0.144	162.2
	3.0	129.0	359.4	0.089	161.0
	3.5	114.2	630.0	0.038	138.3
Dunaliella bardawil	0.5	86.4	549.8	0.011	92.6
(36 days growth)	1.0	137.2	458.5	0.054	161.8
	1.5	164.0	185.8	0.239	208.4
	2.0	186.4	205.9	0.259	239.7
	2.5	203.5	150.2	0.405	264.3
	3.0	202.9	156.5	0.325	253.7
	3.5	215.4	234.9	0.179	257.3
Dunaliella tertiolecta (30 days growth)	0.5	77.84	156.84	0.093	92.43
Dunaliella salina (21 days growth, 97% D2O)	2.0	97.68	14.16	0.038	103.09

Figure 1. Adaptation of Dunaliella tertiolecta to heavy water.

Figure 2. Growth curves of Dunaliella species at their optimal salt concentrations

in H₂O and D₂O.

Figure 3. Growth curve for Dunaliella bardawil in (A) H₂O and (B) D₂O. Data represent mean values of three samples.