Remediation of anthropogenic radionuclides
Tomo Suzuki-Muresan1*, Danis Abdillahi1, Tatsuki Kimura2, Olivier Péron1, Thierry
Lebeau3, Abdesselam Abdelouas1, Fuminori Sakamoto4*, Naofumi Kozai4, Kazuya
Tanaka4, Bernd Grambow1, Keiko Yamaji5***, Misaki Hoshi5, Hayato Masuya6, Haruka
Kato5, Ayako Hoshino5, Aoi Nikkesi5
1 SUBATECH, Unité Mixte de Recherche 6457, École des Mines de Nantes, CNRS/IN2P3,

Université de Nantes, BP 20722, 44307 Nantes cedex 3 France
2 Kyoto University Graduate Scholl of Engineering, Department of Environmental

Engineering, University of Kyoto
3 LUNAM University, LPGN, UMR 6112, BP 92208, 44322 Nantes, France
4 Advanced Science Research Center, Japan Atomic Energy Agency, Japan
5 Graduate School of Life and Environmental Sciences, University of Tsukuba
6 Tohoku Research Center, Forestry and Forest Products Research Institute* Corresponding author: suzuki@subatech.in2p3.fr** Corresponding author: sakamoto.fuminori@jaea.go.jp*** Corresponding author: yamaji.keiko.fp@u.tsukuba.ac.jp

Abstract
New biological remediation methods of radio-Cs doped in simulated contaminated soil
are studied. Three experiments have been conducted: (experiment 1) study on thebio-,phyto-extraction of Cs from doped illite; (experiment 2) study on the development ofaccumulation technique of 137Cs using mushroom mycelium and minerals; (experiment 3)
study on the effect of

137Cs

on symbiosis between Clethra barbinervis androot-endophytes under heavy-metal stress .

1 General introduction
Contamination by actinides (ANs) and fission products (FPs), collectively denoted asanthropogenic radionuclides (ARs), are of great concerns in many countries. For instance,
the disposal of legacy wastes and the decontamination of polluted soils by ARs is acentral issue in the US DOE sites such as the Hanford site [Hartman et al., 2006]. This
site was established to produce military Pu and is today under environmental monitoring.
Hanford site is faced to soil and groundwater contamination by 3H, 129I, 99Tc… US DOE
has developed a large program of cleanup to remediate and monitor the groundwater,
and to reduce the contaminated zone. In Japan, the decontamination of polluted
environments by ARs, mostly radioactive cesium, and the management and the disposalof excavated soils are emerging problems after the accident of the Fukushima Daiichi
nuclear plant. In France the major public health challenges and environmental issues aswell as the potential release of ARs from repository sites in environment for the wastes
management are strongly examined.
Various decontamination strategies have been developed in general, including excavation,
chemical leaching, bio-remediation, and phyto-remediation. Among them, bio- andphyto-remediation are attractive, as they are less invasive against surrounding
environments. ARs readily react with solid phase in soil and rock and change theirchemical forms. For instance, plutonium under reducing and environmental conditions is
expected to be under the form of PuO2 and immobile in soil. However, the presence ofminerals (inorganic material), natural organic materials, microorganisms, and colloids
may initiate the remobilization and its transportation within the environment [Mahara
et al., 2001]. The contribution of micro-organisms on the decontamination of soils needs
to be explored, notably when they are coupled with the phyto-extraction.
Therefore, the objective of the research is to find a new remediation scheme coupling bioand

phyto-remediation

for

soils

contaminated

by

ARs.

Variousbio-andphyto-remediation techniques have been proposed; however, examples of such coupled
schemes are scarce. This new technique could be a cheap and less invasive
decontamination strategy, as similar mechanisms are widespread in nature, whereplants absorb nutrients dissolved from soil minerals, using organic ligands secreted fromtheir roots or coexisting microorganisms.
This work was structured within three parts:–

The bio-,phyto-extraction of Cs from doped illite,–

Development of accumulation technic of 137Cs using mushroom mycelium andminerals,–

Effect of 137Cs on symbiosis between Clethra barbinervis and root-endophytes
under heavy-metal stress

2 Bio‐,phyto‐extraction of Cs spiked‐illite
Biologicalremediation,

such

as

naturalattenuation,

bioaugmentation

andphytoremediation, are used as friendly, cheap and low cost method to decontaminate
polluted soils [Mirsal, 2008]. Bioaugmentation is based on the introduction of
microorganisms in soil, and phytoremediation on the extraction of pollutants by a plant.
Bioaugmentation assisted phytoremediation is a promising method for decontamination
thanks to its synergic effect on mobilization of pollutant in soil directly accessible to theroot of the plants and/or bioavailable in the rhizosphere [Lebeau et al., 2008; Sessitsch etal., 2013].
This clean-up method has given evidence for the decontamination of soil contaminated byheavy metals and petroleum hydrocarbons [Agnello et al., 2016; Lebeau et al., 2008].
This technique was tested to remove cesium from illite, considered as the major mineralclayey phase sorbing Cs, before applying on contaminated soil of Fukushima [Hazotte,
2016; Hazotte et al., 2016]. These authors performed bioaugmentation assisted
phytoremediation experiments in presence of purified siderophores, added up to 250µmolL-1 in contact with Cs-spiked illite with 10 and 100 mmolkg-1 [Hazotte, 2016]. A
partnership Red Clover (Trifolium pratense) – Pyoverdine was put in direct contact with
Cs-spiked soil in pot. The results showed a weak uptake of Cs, up to 1% in the aerial
parts and 0.6% in the roots which was not enhanced with pyoverdine. The expected
efficiency of the method of bioaugmentation assisted phytoremediation is not reachedand the hypothesis of the phyto-toxicity of PVD regarding the roots of the Red Clover israised.
To verify this hypothesis, experiments are performed in this work based on thecompartmentalization of Cs-spiked illite in contact with PVD enclosed in a dialysis
membrane, the contact between PVD and plant being avoided, no Cs is able to diffuse
across the membrane. Three main ongoing experiments are performed (Figure 1):

a. Test of Cs and PVD diffusion through the membrane of dialysis (Figure 1-1)
b. Desorption of Cs from spiked-illite in absence and presence of PVD by Red Clover
phyto-extraction (Figure 1-2)

c. Desorption of Cs from spiked-illite in presence of PVD by KCFC (Figure 1-3)

(1)Cs fully
bioavailable

PVD free inmembrane

Spiked illitewith Cs

Phyto Extraction (2)Extraction by(3)
KCFC (control)

KCFC

Test the propertyof the dialysis
membrane

Spiked illitewith Cs +PVD

Figure 1: Principle of bioaugmentation assisted phytoremediation of Cs from spiked illite
experiment.

2.1

Cesium doped illite

Prior starting the experiments, a stock solution of illite (100 gL-1) are prepared. Purified
illite, kindly provided by BRGM, is put in contact with 100 mmol Cskg-1 of Illite. Aliquots
are measured and almost 80% of Cs is adsorbed onto illite. This suspension is sterilized
by tyndalisation method (3 cycles of 60 min at 70 °C, with 24 h of cooling at ambient
temperature between each cycle) and constitutes the initial stock solution for thebioaugmentation assisted phytoremediation experiments.

2.2

Dialysis membrane performance

Two types of dialysis membrane from Spectrum Lab were tested: Spectra/Por 7 (c.o. 1000
Da) and Spectra/Por Float-A-Lyser® G2 (c.o. 100-500 Da). Their efficiency was assessedin terms of:Resistance against sterilization

Good diffusion of Cs through the membrane

Good retention of PVD inside the membrane

Spectra/Por 7 membrane presents a good resistance against the sterilization by autoclave

(121°C, 20 min) and by tyndalisation. No influence on the diffusion of Cs through themembrane was observed. However, the PVD is not retained by the membrane anddiffuses into the solution, independently of the sterilization method (Figure 2). Therefore, a second dialysis membrane, sterilized Float-A-Lyzer and ready touse dialysis device, was selected to test its performance on the retention of PVD inside
the membrane (Figure 3). The new membrane confines the PVD: no diffusion throughthe membrane was observed. In addition, 100% of cesium diffuse through the membraneamong which 6% of diffused Cs are retained on the floatation ring.

controlTyndalisation @70°C

Autoclave @121°C

PVD

PVD

PVD

PVD

Figure 2: Diffusion test of PVD through Spectrapor7 membrane. Comparison between
tyndalisation and autoclave sterilization method.

(b)

(a)

Figure 3: (a) Caracteristics of the Spectra/Por Float-A-Lyser® G2, (b) Diffusion test of PVD
through Float-A-Lyser membrane (3 repetitions: A, B, C).

2.3

Plant growth

The culture of Red Clover (Trifolium Pratense) is performed in plant growth enclosure.
The growth of plants are performed during 45 days at least and kept in adapted culture
media until their use for the bio-,phyto-extraction experiments.

Figure 4: Growth of Red Clover (Trifolium Pratense) in phytotron.

2.4

Ion exchanger resin

KCFC (potassium cobalt ferrocyanide) is an ion exchanger resin which retains
specifically Cs. KCFC is cleanded, washed and conditioned in NaCl 1×10-3 M. The pH
value of this suspension is adjusted at 8.
As shown in Figure 1-3, KCFC is used to extract selectively Cs during the desorption ofCs from illite by PVD. The aim of this experiment is to control only the efficiency of the
PVD on the desorption of Cs doped onto illite. KCFC is used as a sink for cesium andreplace the role of plants during the extraction process.

2.5

Summary

Property tests on dialysis membrane of Spectr/Por Loat-A-Lyser® G2 are realized which
presents good properties in terms of retention of PVD within the membrane and diffusionof Cs through it. All the elements for the bio-,phyto-extraction are prepared and ready togo: plants come to maturity, sterilized illite doped with Cs, cleaned and conditioned
KCFC. The experiments are ongoing to verify the hypothesis of the phyto-toxicity of PVD
regarding the roots of the Red Clover.

3 Development of accumulation technique of 137Cs using mushroom mycelium
and minerals
A large quantity of radioactive material was released to the environment from Fukushima
Daiichi Nuclear Power Station due to the 2011 Tohoku earthquake and tsunami.
Radioactive materials with short half-lives were serious problem for a few months after
the accident. At the present time after more than six years from the accident, theradioactive materials with medium- and long-half-lives are dominant. Especially, 137Cs
with 30-year half-life gives various limitations to the residents’ life. The decontaminationof living zone such as residential district, public facilities, and cultivated land has been
gradually carried out. However, the decontamination of the forest area remains almost
untouched in Fukushima. The forest area in Fukushima accounts for more than 70% of
the land area of Fukushima prefecture; therefore, in 2016 the Japanese government madea decision to decontaminate the forest areas near residential areas.
It is well known that mushrooms accumulate radioactive Cs. Some mushrooms
accumulate radioactive Cs at high concentration. However, mushrooms can be harvested
only twice (spring and autumn) in one year, and almost all of the mushrooms’ fruit bodies
don’t grow much. On the other hand, fungal mycelia grow all the year except winter. Someof them grow quickly. Therefore, we tried to utilize fungal mycelia to accumulate
radioactive Cs.

Figure 5: The bio-mat set up in the forest

We devised the method to estimate the radioactivity of 137Cs accumulated to mycelium.
The ability of 137Cs accumulation were estimated for about 1500 strains of fungi by using
the method. Then, we made the bag named "bio-mat" containing a fungal mycelium that

accumulates 137Cs efficiently. As a result, we confirmed the bio-mat accumulated 137Cs.
However, as the mycelium died, the accumulated 137Cs began to diffuse to the environment.
Because some minerals stably retain 137Cs, we prepared the new bio-mat containing a
mixture of some minerals. The mineral mixture named "Racoin" is our commerciallyavailable product. It contains zeolite, vermiculite and phlogopite. It was developed toprevent accumulation of 137Cs in mushroom in the first place. The size of the new bio-mat
is a quadrangle of 20 cm x 40 cm. The size of the first bio-mat was a quadrangle of 8 cm x
15 cm. The new bio-mat retained 137Cs for a long period. In the field examination (Figure 5),
we used the new bio-mat without mycelium as a reference. Surprisingly, we confirmed thatthe reference bio-mat also accumulated 137Cs (Figure 6). Some kinds of wild fungi
accumulate 137Cs. Such fungi might have grown in the bio-mat with nutrition andRadioactivity (Bq)accumulated 137Cs.

80
60
40
20
0

Kinds of fungi
Figure 6: Radioactivity of 137Cs in the new biomat containing Racoin.

To confirm the reproducibility, we tried supplementary examination last year. At theexamination, we used the bio-mat of the 50 cm square size. The setting period was up to
150 days. As a result, the following things are suggested; 1) the existence of fungi does not
influence on the accumulation rate of 137Cs, 2) the amount of the accumulated 137Cs
increases with the setting period of the bio-mat, 3) approximately 0.5 weight % of Racoin isenough to add the mat under the present conditions.

4 Effect of 137Cs on symbiosis between Clethra barbinervis and root‐endophytes
under heavy‐metal stressAbstract
Due to the accident at the Fukushima Daiichi nuclear power plant caused by tsunami in
March 2011, large amounts of radioactive materials have been released into
the atmosphere. Especially,

137Cs

contamination might have serious influences onenvironments because its half-life (30.2 years) is longer than other radionuclides. It has
been reported that soil-fungal community have been altered by ionizing radiation,
leading simpler community structure, such as high dominance of stress-tolerantmelanized fungi. Generally, fungi play major roles in forest ecosystem and root
endophytes have been demonstrated to alleviate various kinds of environmental stress toplants. Therefore, the change of endophytes community may affect the symbiosis
interaction between root endophytes and plants. Our targeted tree, Clethra barvinervis
Seib.et Zucc is known to be a tolerant tree against the high concentrations of heavy
metals due to the root endophytes, which increase enhance heavy-metal tolerance. The
purpose of this study is to clarify the effect of

137Cs

on species and chemical

characteristics of root endophytes in C. barvinervis.
We isolated root endophytes from C. barvinervis seedlings growing in 6 different study
sites, showing different concentrations of 137Cs and heavy metals in soil. The following 8
species were isolated from C. barvinervis roots growing in all study sites: Acephala,Colletotrichum,

Cryptosporiopsis,

Lachnum,

Leptodontidium,

Phialocephala,

Rhizoscyphus, and Rhizodermea. We analyzed chemical characteristics of these root
endophytes, such as production of Zn-chelating compounds (detoxicants of Zn) andmelanin (detoxicants of heavy metal as well as defensive compounds against radiation).
Finally, we clarified the effect of 137Cs on species and chemical characteristics of root
endophytes using statistical analyses; non-metric multidimensional scaling (nMDS) andgeneralized linear mixed model (GLMM).
nMDS analysis showed that the appearance numbers of root endophytes and theirchemical characteristics were influenced by 137Cs and heavy metal (Cu, Zn, and Pb)
concentrations in root-zone soil. In contrast, according to GLMM analysis, theappearance numbers of each fungal species were not significantly influenced by 137Cs andheavy metal. GLMM analysis also clarified that Zn-chelating compounds production by

Colletotrichum or melanin production by Acephala showed significantly positive
correlation with 137Cs concentration in root-zone soil. Our results indicate that 137Cs
would influence microflora of root endophytes in C. barbinervis, but in mine sites, themicroflora would be mainly influenced by heavy metals. Additionally, microbial
metabolism related to heavy metal tolerance mechanism would be enhanced by 137Cs inColletotrichum and Acephala species. In conclusion, it suggests that 137Cs would not
influence remarkably symbiosis between C. barbinervis and root endophytes underheavy-metal stress.

4.1

Research Objectives

Due to the accident at the Fukushima Daiichi nuclear power plant caused by tsunami in
March

2011,

large

amounts

the atmosphere. Especially,

137Cs

of

radioactive

materials

havecontamination might havebeen

releasedinto

serious influences onenvironments because its half-life (30.2 years) is longer than other radionuclides. It has beenreported that soil-fungal community have been altered by ionizing radiation, leading simpler
community structure, such as high dominance of stress-tolerant melanized fungi by several
researches after the Chernobyl Nuclear Power Plant accident [Zhdanova et al., 2004;
Zhdanova et al., 1994; Zhdanova et al., 2000]. Generally, fungi play major roles in forest
ecosystem and root endophytes have been demonstrated to alleviate various kinds ofenvironmental stress to plants [Mandyam and Jumpponen, 2005; Rodriguez et al., 2009].
Therefore, the change of endophytes community may affect the symbiosis interaction
between root endophytes and plants. Our targeted tree, Clethra barvinervis Seib. et Zucc is
known to be endemic tree species in Japan, to be observed in old mine sites [Hiroi, 1974] andto be a tolerant tree against the high concentrations of heavy metals due to the root
endophytes, which increase enhance heavy-metal tolerance [Yamaji et al., 2016]. The purposeof this study is to clarify the effect of 137Cs on species and chemical characteristics of root
endophytes in C. barvinervis.

4.2

Research Contents

4.2.1

Elemental analysis of plants and root-zone soil

Five to ten year-old C. barvinervis seedlings (10 seedlings per each site) were collectedfrom 6 different study sites, showing different concentrations of 137Cs and heavy metals
in soil (collection time; July-September, 2012-2015). Zn, Cu, Ni, Pb and Cd in air-driedroot-zone soil (50 x 50 x 50 mm) were analyzed by ICP-OES after acid-digestion,
HNO3-HClO4 (1:4 v/v). Plant materials carefully washed with running water anddeionized water, were separately dried. After the ground materials were pyrolyzed inconcentrated HNO3, heavy metals as described above were quantified by ICP-OES. 137Cs
in the root-zone soil and plants were analyzed with Ge semiconductor detector before theacid digestion. These data were used for the statistical analysis.

4.2.2

Root endophytes isolation

Roots were sterilized using 70% ethanol and 15% hydrogen peroxide. 1,500 root pieces
per each site were incubated on 1% malt extract agar for one month. Fungal detection
rate (%) was calculated using the following formula; Detection rate (%) =Nd/Nt x 100. Nd;
the number of root pieces from which the fungus was detected. Nt; total root pieces forthe isolation. Identification of fungal species were done by DNA analysis as well as
microscopic observation.

4.2.3

Production of Zn-chelating compounds (detoxicants of Zn) by root endophytes

Because Zn accumulation was highly found in C. barbinervis roots [Yamaji et al., 2016],

C. barvinervis would be tolerant to high concentration of Zn. As chemical characteristicsof root endophytes, we considered that root endophytes might produce Zn-chelating
compounds to detoxify Zn in plant tissues or in the rhizosphere [Gadd, 1993]. Seven
fungal species were used for this production test; Rhizodermea, Rhizoscyphus, Lachnum,

Colletotrichum, Cryptosporiopsis, Phialocephala, and Acephala. Six strains per eachfungal species from each study site were used (36 strains per each fungus in total).
According to [Martino et al., 2003], insoluble ZnO and Zn3(PO4) were mixed with medium
and root endophytes were inoculated onto the medium. After 7-days incubation, clear
zone was formed by Zn-chelating compounds on the medium. Zn-chelating activity wascalculated using the following formula; Zn-chelating activity =(Cd-Ed)/Ed. Cd; thediameter of clear zone. Ed; the diameter of root endophytes.

4.2.4

Production of melanin by root endophytes

Melanin is known to be detoxicants of heavy metal [Martino et al., 2003] as well as
defensive compounds against radiation [Dadachova and Casadevall, 2008]. The following
7 fungal species were used; Rhizodermea, Rhizoscyphus, Lachnum, Colletotrichum,

Cryptosporiopsis, Phialocephala, and Acephala (same strains were used in 2.2).
Root endophytes grown on potato-dextrose agar for 3 weeks were used for melanin
extraction with 1M NaOH [Gadd, 1982]. According to [Gadd, 1982], melanin production
was measured at 405 nm.

4.2.5

Statistical analysis

Clarification of the effect of 137Cs on microflora and chemical characteristics of root
endophytes using non-metric multidimensional scaling (nMDS) and generalized linear
mixed model (GLMM). the vegan package of R [Oksanen et al., 2015] with 10000
permutations was used to examine differences.

4.3

Research results

4.3.1

Root endophytes isolation

The following 8 species were isolated from C. barvinervis roots growing in all study sites:

Acephala, Colletotrichum, Cryptosporiopsis, Lachnum, Leptodontidium, Phialocephala,
Rhizoscyphus, and Rhizodermea. These root endophytes were used for the production
test of Zn-chelating compounds.

4.3.2

Production of Zn-chelating compounds and melanin by root endophytes

Results of Zn-chelating compounds production were shown in Fig. 1. Colletotrichum,

Cryptosporiopsis and Acephala isolated from all sites produced Zn-chelating compounds.
Rhizoscyphus, Lachnum and Phialocephala did not show the production. Results ofmelanin were shown in Fig. 2. All strains of Acephala, Phialocephala, Cryptosporiopsis
and Colletotrichum, and several strains of Rhizodermea

produced melain.

Rhizoscyphus and Lachnum produce very small amounts of melanin. Strains show
different patterns of melanin production even among each fungal species.

Fig. 1 Production ability of Zn-chelating compounds by root endophytesFig. 2 Production ability of melanin by root endophytes

Data was shown as average ± SE. Different colors means different study area; blue,
brown and white bar graphs indicate old mine sites (high concentrations of heavy metals).
Blue and white bar graphs also indicate moderate concentrations of 137Cs. Yellow andorange bar graphs indicate Fukushima study sites, which show high concentrations of
137Cs. Green bar graphs means natural forest, which did not contain high concentrationsof 137Cs and heavy metals.
Data was shown as average ± SE. Different colors means different study area; blue,
brown and white bar graphs indicate old mine sites (high concentrations of heavy metals).
Blue and white bar graphs also indicate moderate concentrations of 137Cs. Yellow andorange bar graphs indicate Fukushima study sites, which show high concentrations of
137Cs. Green bar graphs means natural forest, which did not contain high concentrationsof 137Cs and heavy metals.

4.3.3

Statistical analysis

nMDS clarified 137Cs and heavy metals (Cu, Zn, and Pb) in the root-zone soil influence
the microflora of root endophytes (Fig. 3). Microflora in mine sites (red circle; Fig. 3)
would be influenced by mainly heavy metals. Chemical characteristics of root endophytes
would be also influenced by these environmental factors (data not shown). GLMM
analysis clarified that 137Cs induces productions of Zn-detoxicants and melanin. For
example, production of Zn-chelating compounds by Colletotrichum strains showed
positive correlation with 137Cs in root-zone soil (p<0.05). Production of melanin by

Acephala strains showed positive correlation with 137Cs in root-zone soil (p<0.05).

Fig. 3 Result of nMDS analysis

Red, yellow and orange spots mean mine sites. Blue and indigo blue spots indicate
Fukushima study sites, which show high concentrations of 137Cs.Green spots meannatural forest, which did not contain high concentrations of 137Cs and heavy metals.

4.4

Conclusion

137Cs would influence microflora of root endophytes in

C. barbinervis, but in mine sites,

the microflora would be mainly influenced by heavy metals. Microbial metabolism
related to heavy metal tolerance would be induced by 137Cs in Colletotrichum andAcephala species. Under mine sites together with radio Cs, heavy-metal stress toleranceof C. barbinervis enhanced by root endophytes would be maintained.

5 Acknowledgement
The authors acknowledge the financial support from REIMEI project. We would like toaddress our sincere gratitude to Nolann Longeard from Subatech laboratory forexperimental tests.

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