Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 99-101, 2005

Accepted
Effect of Uranium (VI) on the Growth of Yeast and Influence of Metabolism of Yeast on
Adsorption of U (VI)
F. Sakamoto,*, a T. Ohnuki,a N. Kozai,b E. Wakai,c T. Fujii,d H. Iefuji,d and A. J. Francise
aAdvanced Science Research Center, Japan Atomic Energy Research Institute, Shirakata-2, Tokai, Ibaraki 3191195, Japanb
Department of Environmental Sciences, Japan Atomic Energy Research Institute, Shirakata-2, Tokai, Ibaraki319-1195, Japanc
Department of Materials Science, Japan Atomic Energy Research Institute, Shirakata-2, Tokai, Ibaraki 3191195, JapandEnvironmental Research Division, National Research Institute of Brewing, Higashi Hiroshima 739-0046, Japane
Environmental Sciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA
Received: November 15, 2004; In Final Form: April 21, 2005
We have carried out the growth experiments of 3 strains of yeast in a medium containing uranium (VI) to elucidate the effect of U (VI) on the growth of microorganisms. Hansenula fabianii J640 grew in the liquid mediumcontaining 0.1 mM U (VI) at lower rate than the control, but Saccharomyces cerevisiae did not grow under thiscondition. The H. fabianii J640 pre-cultured for 21 h in the liquid medium without U (VI) grew even after theexposure to 1 mM U (VI), but did not grow without pre-cultivation. For the pre-cultured H. fabianii J640,
radioactivity of U in the medium was the same as the initial one for 110 h, and then gradually decreased. TEMEDS analysis of H. fabianii J640 exposed to 1 mM U (VI) for 165 h showed accumulation of U (VI) on the cells.
When H. fabianii J640 was not pre-cultured, radioactivity of U in the medium was lower than the initial one.
These results indicated that U (VI) inhibits the growth of yeast, and that the adsorption of U (VI) by the cells
depends on the metabolism of yeast.

1. Introduction
The contamination of the environment by uranium frommine tailing and U mine water is major environmental concern
because of α-radioactivity. It is well known that microorganism accumulates U1−3 and microorganism plays a key role
in regulating the mobility of uranium in the underground.4−6
Many researchers, therefore, have extensively studied theinteraction of microorganism with U.7−10 They suggested thatmicroorganism may be utilized to immobilize U from U minetailings, U mine wastewater and radioactive waste fromnuclear reactors.
Leduc et al.7 reported that U was twelve times more toxic
than copper and nickel for Thiobacillus ferrooxidans. Suzukiand Banfield studied the toxicity of U to microorganisms.11
However, to our knowledge, little is known about effect of U
on the growth of microorganisms.
Resting cells of the yeast Saccharomyces cerevisiae is known
to accumulate U (VI).9, 10 Although the yeast biomass obtained
from brewery waste has been proposed as a treatment process
to remove U from waste stream, the effect of U on the growthof yeast in the liquid medium is poorly understood. In thisstudy, we investigated the effect of U (VI) on the growth ofseveral strains of yeast.
2. Experimental
In order to examine the effect of U on growth we tested 31
strains of yeast including Saccharomyces cerevisiae X-21801B, Hansenula fabianii J640, and Hansenula anomala J224.
These yeasts included 7 types of brewer’s yeast for ‘sake’, 5types of brewer’s yeast for beer, 3 strains of brewer’s yeast for*Corresponding author. E-mail: buntoku@popsvr.tokai.jaeri.go.jp.
FAX: +81-29-282-5927.

wine, 4 strains of brewer’s yeast for Japanese spirits, and 2
strains of baker’s yeast.
The yeasts were grown in YNB medium consisted of 5.7 gof YNB without Phosphate (Qbiogene, Inc.), 20 g of glucose
and 100 mg of β-glycerophosphate per liter. Solid media
contained 2% Bacto-agar (BD Biosciences). UO2(NO3)2 wasdissolved in deionized water and the concentration wasadjusted at 100 mM.
The yeasts were cultured on YNB agar media containing
0.1 mM or 1.0 mM U (VI) for 72 h at 30 ˚C. For further investigation, S. cerevisiae X-2180-1B and H. fabianii J640 werecultured in 100 mL of YNB liquid media containing 0 (control),0.1, or 1.0 mM U (VI) up to 165 h at 30 ˚C. The initial amountof yeast in the media was adjusted to 0.1 at the optical density
at 600 nm (OD600).
In order to examine the effect of pre-culture of yeast, H.
fabianii J640 was cultured in YNB liquid for 21 h at 30 ˚Cfollowing the exposure to U (VI). The growth of the yeast inthe liquid media was estimated by measuring OD600 at 21, 48,70, 110, 140, and 168 h after the inoculation. U concentrationsin the media were measured by radiometry using liquid scintillation counter at 21, 48, 70, 110, 140, and 165 h. The cells ofH. fabianii J640 exposed to 1 mM U (VI) were analyzed bytransmission electron microscopy (TEM) (Hitachi HF-2000)
operating at 200 kV. Elemental analysis was carried out by
EDS attached to the TEM using a Kevex Sigma system software package. A small amount of sample of the wet precipitates was dropped on a copper grid sample folder for TEM
observation. The sample was dried for 24 h in a desiccator.
3. Results and Discussion
H. fabianii J640 and H. anomala J224 showed a high tolerance for U (VI) among yeast strains tested. They grew on YNB
agar medium containing 1 mM U (VI), while S. cerevisiae X-© 2005 The Japan Society of Nuclear and Radiochemical SciencesPublished on Web 07/31/2005

Sakamoto

100 J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

No U (control)

Optical density at 600 nm

1080.1 mM U(VI)64

concentration of U (VI) added. This suggests that some fractions of U (VI) were sorbed by the cells of H. fabianii J640. The
inhibition of the growth of H. fabianii J640 is probably causedby the sorption of U (VI).
On the other hand, the radioactivity in the medium for thepre-cultured sample was approximately 350 cpm and it
remained constant up to 72 h, whereas the OD600 was reached
to its maximum from 2 to 5. The radioactivity then decreased
with time (Figure 3). TEM image of whole mounts of H. fabianii J640 cells at 164 h (Figure 4) showed clear contrast
without further staining, suggesting that U (VI) was accumulated by the J640 cells. Santamaria et al.12 pointed out that theeffect Th on Bradyrhizobium growth was reduced by theOptical density at 600 nm

10No U (control)

8Pre-culture64

Non-pre-culture
20Pre-culture
Non-pre-culture

300

Radioactivity (cpm)

2180-1B did not.
Figures 1 and 2 show the growth curves of H. fabianii J640
and S. cerevisiae X-2180-1B, respectively, cultured in YNB
liquid media containing 0.1 mM and 1 mM U (VI). H. fabianii
J640 grew in the liquid media containing 0.1 mM U (VI),
while S. cerevisiae X-2180-1B did not. The OD600 of H. fabianii J640 grown in the liquid media containing 0.1 mM U (VI)was lower than that of control. The OD 600 reached a steadyvalue of approximately 5 at 48 h after inoculation in themedium containing 0.1 mM U (VI). In contrast, the OD600 ofcontrol sample increased with time up to 164 h. These results
indicated that U (VI) inhibited the growth of H. fabianii J640.
Suzuki and Banfield11 reported that the radioactivity of U was
not lethal to microorganisms, but the chemical properties of U
caused significant toxic effects.
Neither H. fabianii J640 nor S. cerevisiae X-2180-1B grewin the liquid medium containing 1 mM U (VI). On the otherhand, H. fabianii J640 grew up to about 5 of OD600 (the upperpanel of Figure 3) in the medium containing 1 mM U (VI)after the pre-cultivation in the liquid media without U (VI) for
21 h. H. fabianii J640 grew to 2 of OD600 for 21 h. Additionof 1 mM U (VI) to the culture at 21 h resulted in an increase inOD600 to 5 at 50 h. However, no significant increase in OD600
was observed for the culture after 50 h in comparison to thecontrol sample without U (VI) exposure.
Radioactivity of U in the liquid media containing 1 mM U(VI) exposed to H. fabianii J640 was approximately constantat 250 cpm (the lower panel of Figure 3). This value was lower
by about 0.7 than that of the radioactivity estimated by the initial

200

100001 mM U(VI)2005010050100150

Culture time (hours)
Figure 3. Growth of H. fabianii J640 in YNB liquid medium
containing 1 mM U (VI) (upper) and radioactivity of U (VI) in theliquid medium (lower).

150

Culture time (hour)
Figure 1. Growth of H. fabianii J640 in YNB liquid medium
containing 0.1 mM or 1 mM U (VI).

Optical density at 600 nm

2No U (control)

1:0.1 mM U(VI):1 mM U(VI)0050100

150

Culture time (hour)Figure 2. Growth of S. cerevisiae X-2180-1B in YNB liquid medium
containing 0.1 mM or 1 mM U (VI).

Figure 4. TEM image of 140-h cells of H. fabianii J640 pre-cultured
for 21 h followed by incubation with 1 mM U (VI).

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005 101

Effect of Uranium (VI) on the Growth of Yeast and Influence

precipitation of Th. We assume that the accumulation of U(VI) by yeast cells is a different phenomenon from the Th
precipitation observed in Bradyrhizobium.
Volesky et al.9 and Strandberg et al.10 reported the resting
cells of S. cerevisiae adsorbed U for several hours of incubation. These results are in agreement with the sorption of U(VI) by H. fabianii J640 without pre-culture. However, whenH. fabianii J640 was pre-cultured, the adsorption was notobserved up to 72 h. This suggests that the accumulation
behavior of U (VI) under the pre-culture condition is different
from the resting conditions. Francis et al. 13 indicated thatcarbonate produced by the metabolism of bacteria dissolved U(VI)-phosphate precipitates. At 21 h, the carbonate concentration should be high in the pre-culture due the growth of H.
fabianii J640, even though the carbonate concentration was notmeasured. U (VI)-carbonate complex has low sorption ability
on minerals and microorganisms. Thus, the effect of U (VI) on
the growth of H. fabianii J640 may be masked by carbonate
produced at the initial stage of the exposure to U (VI).
However, at the late stage, where OD600 was saturated atapproximately 5 (Figure 3), metabolic activity of H. fabianii
J640 probably lower than the initial stage, and the decreased
concentration of carbonate resulted in the sorption of U (VI)on the cell surfaces of H. fabianii J640.
4. Conclusions
The yeasts H. fabianii J640 and H. anomala J224 grew inYNB agar medium containing 1 mM U (VI), though S. cerevisiae X-2180-1B did not. H. fabianii J640 grew in YNB
liquid medium containing 0.1 mM U (VI), though S. cerevisiae
X-2180-1B did not. This indicates that H. fabianii J640 has
higher tolerance to U (VI) than S. cerevisiae X-2180-1B.
Neither H. fabianii J640 nor S. cerevisiae X-2180-1B grew inthe liquid media containing 1 mM U (VI). However, after precultivation, H. fabianii J640 grew up to about 5 of OD600 in theliquid media even containing 1 mM U (VI). According to
TEM observation, U was present on the cells of the yeast.
These results suggest that the metabolic products of the yeast
affect the adsorption of U to the cells.
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