Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 21-24, 2005

Accepted
Microbial Influences on the Mobility and Transformation of Radioactive Iodine in theEnvironment
Seigo Amachi,*, a Takaaki Fujii,a Hirofumi Shinoyama,a and Yasuyuki MuramatsubaDepartment of Bioresources Chemistry, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271-8510, Japan
Department of Chemistry, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japanb

Received: November 15, 2004; In Final Form: April 21, 2005
Long-lived radioactive iodine (129I, half-life: 1.57 × 107 y) has been released into the environment from nuclear
fuel reprocessing plants. 129I may also be released from ground storage of nuclear waste. Given its long half-life,
a better understanding of the behavior of iodine in the environment is necessary to ensure the safety of humans
and the health of the environment. In this report, we summarize our recent results and new experimental data
about microbial influences on the mobility and transformation of iodine. Microbial volatilization of organic
iodine was observed in soil slurries and seawater samples, and various species of aerobic bacteria were considered
to play a significant role through methylation of iodide (I −) to form methyl iodide (CH3I). The volatilization ofiodine was also found in iodide-rich natural gas brine water, where iodide concentration is approximately 2,000
times higher than that in seawater. In this case, however, a significant amount of molecular iodine (I 2) was
produced together with organic iodine compounds. Iodide-oxidizing bacteria, which oxidize iodide to I2, wereisolated from seawater and natural gas brine water. Phylogenetically, they were divided into two groups within
the α-subclass of the Proteobacteria (Roseovarius sp. and unidentified bacteria), and they produced not only I2 but
also diiodomethane (CH2I2) and chloroiodomethane (CH2ClI). Iodide-accumulating bacteria, which accumulate
iodide to concentrations 5,500-fold over that of the medium, were also isolated from marine sediment. They wereclosely related to Arenibacter troitsensis, and iodide uptake was mediated by an active transport system. Our
results suggest that the fate of iodine can be affected by microorganisms, particularly by bacteria, throughprocesses such as volatilization, oxidation, and accumulation.

1. Introduction
Iodine-129 (129I, half-life: 1.57 × 107 y) is one of the most
persistent anthropogenic radionuclides, and it has beenreleased into the environment from nuclear weapons testingand nuclear fuel reprocessing plants.1, 2 129 I has the same
geochemical properties as stable iodine (127I), and is rapidly
dispersed in the environment since its predominant chemical
forms iodide (I − ) and iodate (IO 3 − ) are highly soluble andmobile. In the UNSCEAR-2000 Report,3 129I is regarded as
one of the most critical radionuclides to be assessed from theviewpoint of global circulation. Therefore, a better understanding of the behavior of iodine throughout the biosphere isneeded to ensure the safety of 129I. However, the biogeochemical cycling of iodine is not fully understood. Since microorganisms affect the mobility and speciation of many traceelements in the environment,4 it is possible that the chemical
behavior of iodine is also influenced by microbial activities. In
this paper, we summarize our recent investigations and present
new experimental data in relation to microbial effects on thechemical behavior of iodine in the environment.
2. Experimental
Volatilization of Iodine from Environmental Samples.
Soil slurries, fresh seawater, and natural gas brine water weredispensed into 120-mL serum glass bottles, and radioactive
iodide tracer (125I −) was added to give a final concentration of
approximately 30 kBq/mL. In order to change the composition
and the level of microbial populations, yeast extract, cycloheximide (as a eukaryotic antibiotic), or a mixture of streptomycin*Corresponding author. E-mail: amachi@faculty.chiba-u.jp. FAX:
+81-47-308-8866.

and tetracycline (as prokaryotic antibiotics) was added to somesamples to give final concentrations of 1 g/L, 0.15 g/L, and0.025 g/L each, respectively. The final volume of the samples
was adjusted to 20 mL. The bottles were sealed with butyl
rubber stoppers, and incubated for 2 to 7 days with shaking.
Volatile organic and inorganic iodine compounds werecollected with activated charcoal and silver wool traps, respectively.5 The activity of 125I was measured with a NaI scintillation counter (Aloka ARC-370M). In some cases, chemical
formulas of the organic iodine compounds were identified by agas chromatograph equipped with an electron-capture detector(GC-ECD) and GC coupled to a mass-selective detector (GCMS).
Experiments with Bacterial-Pure Cultures. Bacterial
capacities for volatilizing iodide were determined by radiotracer
techniques. Bacterial strains, isolated from soil, seawater,
marine sediment, and marine algae, were cultured with 125I − inserum glass bottles. Volatile organic iodine compounds werecollected and determined as described above. The strains were
judged to have abilities to volatilize iodide when their percentages of volatilization were more than 0.01%. In some cases,
chemical formulas of the organic iodine compounds emitted
from the cultures were identified by GC-ECD and GC-MS.
To isolate iodide-oxidizing bacteria, soil, seawater, marine
sediment, and natural gas brine water were diluted and spreadon agar media containing stable iodide (KI) and soluble
starch.6 In some cases, environmental samples were enrichedwith 1 mM of iodide for 7 to 180 days before they were spreadon the media. If iodide was oxidized to I 2 by bacteria, thecolor of the bacterial colonies became purple since I2 forms a
purple complex with starch. Bacterial production of I2 andorganic iodine compound was determined by radiotracer techniques. Chemical formulas of the organic iodine compounds
were also identified by GC-MS. The iodide-oxidizing enzyme© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

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AmachiJ. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

activity was assayed spectrophotometrically using the culture
supernatant as a crude enzyme.
For isolation of iodide-accumulating bacteria, two types ofscreening were carried out.7 In the first screening, various bacterial
strains maintained in our laboratory were inoculated onto agar
media containing 125 I −. After incubation, bacterial colonies weretransferred to a membrane filter by toothpicks, and radioactivity in the colonies was visualized with a Molecular Imager
FX system (Bio-Rad). This transfer technique enabled us to obtaina good autoradiograph with a relatively low background, while
direct transfer of colonies without toothpicks resulted in a very
high background. Bacterial strains showing dense and black spots
were chosen. In the second screening, the strains were cultured inliquid media containing 125 I −. After incubation, the cultures wereput on a silicone oil layer (35:65 mixture of SH556 and SH550,
Toray Dow Corning Silicone) that was overlaid on distilled waterin a microcentrifuge tube. After centrifugation, the tube contents
were frozen, and the distilled water layer containing the cell pellet
was cut off for determining radioactivity.
For phylogenetic analysis of bacterial strains, the 16S ribosomal
RNA gene (16S rDNA) was sequenced as described previously.8
The sequences were aligned using the CLUSTAL W program,version 1.6.9 A phylogenetic tree was constructed by the neighborjoining method.10
3. Results and Discussion
Volatilization of Iodide from Environmental Samples.
When diluted soil slurries were incubated with 1 mM of stable
iodide for 20 days, CH3I production in the bottle headspace
was detected by GC-ECD.8 Production of other organic iodine
compounds (e.g. CH3CH2I and CH2I2) was not observed. The
addition of glucose sometimes enhanced CH3I production, butlittle CH3I was produced under anaerobic incubation conditions. The soil slurries were then incubated with iodide tracer(125I−), and iodide volatilization under various incubation conditions was determined. This method allowed us to study iodide
volatilization under a natural concentration of stable iodide(without any addition of excess stable iodide). Autoclaving ofsoil slurries strongly inhibited the volatilization, while yeast
extract sometimes enhanced the volatilization. Interestingly,
the volatilization of iodide was almost completely inhibited byprokaryotic antibiotics (streptomycin and tetracycline), but notby the eukaryotic antibiotic cycloheximide. These results
suggest that iodide in soil is methylated and volatilized into theatmosphere mainly as a result of aerobic soil bacteria.
A similar experiment was carried out using fresh seawater.11
The volatilization of iodide did not occur in autoclaved or 0.22µm-filtered seawater. In addition, both prokaryotic andeukaryotic antibiotics inhibited the volatilization significantly.
Thus, it was considered that iodide in seawater is volatilized asa result of microbial activities, and that marine bacteria, atleast partially, participate in the process.
In soil slurries and seawater, iodide was volatilized mainly as
an organic iodine compound (probably CH3I), and volatilizationof inorganic iodine (i.e. molecular iodine: I2) was not observed.
However, when we incubated natural gas brine water with 125I−,
not only organic iodine but also I2 was produced. Table 1 shows
new experimental data related to the production of I 2 in brinewater. I 2 production was much higher than that of organic
iodine (see “untreated” samples in Table 1), and it was enhancedby the addition of 0.1% yeast extract. The volatilization of I2
was strongly inhibited by autoclaving, filtration, and prokaryotic antibiotics. These results suggest that I 2 production inbrine water is also mediated by bacteria. From the viewpointof global iodine cycling, natural gas brine water is of greatinterest because it is highly enriched with iodide. Brine waterin Japan, especially that in Chiba prefecture, often has iodide
concentrations of approximately 1 mM, which is 2,000 times

12higher than that in natural seawater. Natural gas, mainly
methane, is associated with brine water in this area. The
annual production of iodine in this area accounts for 6,000
tons, which constitutes approximately one third of the world
iodine production. Our results indicate that bacteria withcapacities for producing I 2 and organic iodine compounds
inhabit brine water. Isolation and characterization of suchbacteria is described below (see Iodide-oxidizing Bacteria(IOB)).
TABLE 1: Organic and Inorganic Iodine Volatilizationfrom Natural Gas Brine Waters a
Untreated Autoclaved Filtered b

YE c Stp+Tcd Che

Brine 1 Org-I

100

0

0

943

0

86

Inorg-I266

0

9

10,059049Brine 2 Org-I

100

2

2

211

33

71

Inorg-I

600

0

0

2,781

N.D.f

N.D.

a

All values are expressed as relative % of volatilization, and volatilization of organic iodine in untreated samples is expressed as 100%.
b
Samples were filtered through a 0.22-µm membrane filter. cYeast
extract was added. d Streptomycin and tetracycline were added.
e
Cycloheximide was added. f Not determined.

Iodide-volatilizing Bacteria (IVB). Of 100 bacterial strainstested, approximately 40% showed percentages of volatilization of more than 0.01%.8, 11 Based on 16S rDNA sequences,
we concluded that they were affiliated with Proteobacteria (α,β, and γ subdivisions), the Cytophaga-Flexibacter-Bacteroides(CFB) group, and High G+C Gram positive bacteria, indicating that IVB were not confined to any particular group of
bacteria and were found throughout the bacterial domain. GCECD and GC-MS analyses showed that the gaseous iodine
compound emitted by bacteria was CH3I. We also measured
the iodide-methylating activity by using cell free extracts of
Rhizobium sp. MRCD 19. 13 We found that S-adenosyl- L methionine (SAM) could serve as a methyl donor for methylation of iodide. The apparent affinity constants (K m ) for
substrates (i.e. iodide and SAM), calculated from LineweaverBurk plots, were 0.26 and 0.024 mM, respectively.
Iodide-oxidizing Bacteria (IOB). A large number of IOB
strains were isolated from natural gas brine water, seawater,and marine sediment.6 Interestingly, an enrichment of theenvironmental samples with 1 mM of iodide (7 to 180 days)
was essential for isolation of IOB from seawater and sediment,
while IOB could be isolated without enrichment from brinewater. IOB were phylogenetically divided into two groups
within the α-subclass of the Proteobacteria. One of thegroups was most closely related to Roseovarius tolerans withsequence similarities between 94 and 98%. The other group
was most closely related to Rhodothalassium salexigens,
although the sequence similarities were relatively low (89 to91%). The iodide-oxidizing reaction was mediated by an
extracellular enzyme, and oxygen was indispensable for thereaction. This inferred that certain oxidases were involved in
the oxidation of iodide.
Radiotracer experiments and GC-MS analyses revealed that
IOB produced not only I2 but also organic iodine compounds
such as CH2I2 and CH2ClI.6 Table 2 shows our new experimental data on gaseous iodine production by IVB (strain C-19)
and IOB (strains A6 and C3). IVB did not produce I2, and the
production amounts of organic iodine compound (CH3I) under
0.1 µM and 0.1 mM of iodide were 21 and 730 pmol/20 mL ofculture, respectively. Since the organic iodine production
increased only 35-fold in the presence of 0.1 mM iodide, CH3I
production from iodide was considered to be saturated. In IOB

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

Microbial Influences on the Mobility and Transformation

TABLE 2: Production of Organic and Inorganic Iodine
Compounds by IVB and IOBa

Iodide-accumulating bacterium strain C-21 (AB182553)
53[Flexibacter] aggregans NBRC15975 (AB078039)
100

Arenibacter troitsensis (AB080771)

100

StrainIodide concentration added tothe medium

Most-relatedorganism

0.1 M

0.1 mM

Arenibacter latericius (AF052742)

94

Zobellia uliginosa (M62799)

1 µM

100

55

Zobellia galactanivorans (AF208293)

31

C-19 (IVB) Algoriphagus
antarcticus

Org-I

21

730

N.D.b

I2

<0.04

<40

N.D.

23

Vitellibacter vladivostokensis (AB071382)[Cytophaga] marinoflava (M58770)

44

Cellulophaga baltica (AJ005972)
100

Cellulophaga algicola (AF001366)
88

Cellulophaga fucicola (AJ005973)

A-6 (IOB)

Roseovarius
tolerans

Org-I

0.11

17,000

N.D.

I2

<0.04

840

N.D.

100

45

Cellulophaga lytica (M62796)
Psychroflexus torquis (U85881)
100

Psychroflexus gondwanensis (M92278)
59

94

Salegentibacter salegens (M92279)
54

C-3 (IOB)

Rhodothalassium Org-I
salexigens
I2

<0.04

64,0005,200,000[Cytophaga] latercula (D12665)
Gelidibacter algens (AF001367)

<0.04

1,200

89

160,000

Psychroserpens burtonensis (U62913)
56

Polaribacter filamentus (U73726)a

All values are expressed as pmol of iodine/20 mL of culture. The
strains were cultured with 125I− and appropriate concentrations of stable
iodide (KI) in Marine Broth 2216 liquid medium (Difco). After 3 daysof incubation, volatile organic iodine and I2 were collected with activated charcoal and silver wool traps, respectively. bNot determined.

97
98

Polaribacter glomeratus (M58775)
Polaribacter franzmannii (U14586)

100

Polaribacter irgensii (M61002)
100

Tenacibaculum ovolyticus (AB032506)
94

Tenacibaculum mesophilum (AB032501)

99

Flavobacterium aquatile (M62797)

56
100

Flavobacterium johnsoniae (M59051)

90

Myroides odoratus (M58777)
Capnocytophaga haemolytica(U41349)

0.01

Figure 1. Phylogenetic position of strain C-21 among the family
Flavobacteriaceae on the basis of 16S rDNA sequences. Numbers at
nodes show bootstrap values obtained from 100 resamplings. The
GenBank accession number for each reference strain is shown inparentheses. The bar shows 1 nucleotide substitution per 100
nucleotides. Capnocytophaga haemolytica was used as the outgroup.

10

8

6
V (pmol iodide min -1 mg dry cells-1)

1/V

strains, however, both organic (CH2I2 and CH2ClI) and inorganic (I2) iodine compounds were produced, and the production was strongly enhanced under high levels of iodide (Table2). This may imply that the expression of enzyme(s) that
produced I2, CH2I2, and CH2ClI was induced or enhanced underhigh iodide concentrations. However, in our previous study,6
the levels of iodide-oxidizing activity were constant regardlessof iodide concentrations in the media. Thus, it might bepossible that the activity of the enzyme(s) involved in the
production of I2, CH2I2, and CH2ClI is induced (or enhanced)
by I2 but not by iodide. Further study is needed to understandthe biochemical mechanism of organic and inorganic iodine
production by IOB.
Iodide-accumulating Bacteria (IAB). A screening of bacteria with capacities for accumulating iodide was carried out.7
Combining autoradiography with radiotracer experiment wasvery efficient for selection of IAB. Of 91 bacterial strainstested, 6 strains were chosen as IABs. They removed 80 to
90% of iodide from the liquid media within 24 h, and a corresponding amount of iodide was detected in their cells. All ofthese strains originated from marine sediment, and they were
phylogenetically most closely related to Arenibacter troitsensis
and Flexibacter aggregans NBRC15975, members of the family Flavobacteriaceae. Figure 1 is a phylogenetic tree of strain
C-21, which was chosen for further investigation. When strain
C-21 was grown in a liquid medium containing 0.1 µM of
iodide, iodide concentration in the medium decreased to 0.02
µM within 24 h, and the maximum iodide content of the strain
was 220 pmol/mg dry cells. The iodide concentration factor
was calculated based on the ratio of the iodide concentration incells (on a dry weight basis) to that in the medium. In strain C21, the maximum value of the iodide concentration factor was
5,500 at 24 h. The kinetics of iodide uptake by IAB were thendetermined using a washed cell suspension of strain C-21. A
cumulative uptake of iodide was observed, but it occurred onlyin the presence of glucose. Iodide transport by this strain
showed Michaelis-Menten kinetics with an apparent affinity
constant (Kt ) and a maximum velocity (Vmax) of 0.073 µM and
0.55 pmol/min/mg dry cells, respectively (Figure 2). Fromthese results, we considered that the uptake and accumulationof iodide by IAB was mediated by an active transport system
which requires metabolic energy, but not by simple diffusionor physicochemical adsorption onto the bacterial cell surface.
Determinations of the energy source (ATP or ion gradient) andthe substrate specificity of the iodide uptake system are underway in our laboratory.

Tenacibaculum maritimum (M64629)

4

2

-20

-10

0

10

20

30

0.5
0.4
0.3
0.2
0.1
0
0

0.1

0.2

0.3

0.4

0.5

iodide concentration (µM)

40501/ [iodide] (µM-1)
Figure 2. A double-reciprocal plot of iodide uptake by strain C-21.
The inset shows the initial uptake of iodide as a function of iodide
concentrations from 0.02 to 0.5 µM.

4. Conclusion
Microorganisms play an important role in the biogeochemical cycling of many trace elements by changing their chemical
behavior in the environment. Our results demonstrated thatmicroorganisms, especially bacteria, have a potential to influence the chemical behavior of iodine through such processes asvolatilization, oxidation, and accumulation. Another important
process in iodine cycling is the reduction of iodate (IO 3−) toiodide, and a sulfate-reducing bacterium (Desulfovibrio desul-

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AmachiJ. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

CH2I2, CH2ClICH3IOxidation &Volatilization (IOB)

Volatilization (IVB)

I- (-1)I2 (0)
Reduction(MRB, SRB)?

Accumulation (IAB)

Spontaneous

IO3- (+5)
HOI (+1)

Immobilization

Spontaneous

Figure 3. The biogeochemical cycling of iodine. Pathways that canbe mediated by microorganisms are also shown.

furicans) and a metal-reducing bacterium (Shewanella putrefaciens) have been reported to possess iodate-reducing abilities.14, 15
In Figure 3, we illustrate the biogeochemical cycling of iodine,
and also show several pathways that can be mediated by microbial activities. Since bacteria comprise a great biomass and are
distributed widely in the environment, it is not surprising if they
contribute significantly to global iodine cycling. Therefore,
bacterial influence on the mobility and the transformation ofiodine should be considered in the assessment of 129I released
from nuclear facilities. Furthermore, such information may be
useful in predicting the environmental risk of 129 I possibly
released from ground storage of nuclear waste.
Acknowledgements. We thank S. Yoshida (National Instituteof Radiological Sciences), Y. Kamagata (National Institute ofAdvanced Industrial Science and Technology) and T. Ban-nai(NIRS) for their valuable comments and kind cooperation andsupport throughout this study. We also grateful to M. Kasahara,
Y. Mishima, and Y. Akiyama (Chiba Univ.) for their technicalsupport. This work was partly supported by funds from theForum on Iodine Utilization (FIU) and the Hamaguchi
Foundation for the Advancement of Biochemistry granted to S. A.
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