Enhancement of cesium-ion-uptake function of membrane
vesicles and establishment of ion recovery technology
Masahiro ITO
Faculty of Life Sciences, Toyo University
Research objective
Twelve years after the occurrence of the Fukushima Daiichi Nuclear Power Plant accident, therelease of treated water, comprising the radionuclide-free portion of the ever-increasing
amounts of contaminated water, into the ocean continues to be a focal issue. This is because
in more than 70% of the stored “treated water,” radioactive nuclides such as radioactive
strontium (Sr) have not been removed to levels below the detection limits set for release into
the ocean. In addition, there are problems such as the need to continually perform radionuclide
removal processing to achieve the requisite levels 1). Thus, there is still a need for more
effective and inexpensive techniques for radionuclide removal (particularly Sr and cesium(Cs)) from the contaminated water. Environmental purification technologies using
microorganisms are inexpensive and can maintain a low ecological load 2). Meanwhile, thehalf-life of 137Cs can be as long as 30 years, and long-term environmental pollution can thus
become a significant problem. In addition, Cs has similar physicochemical properties topotassium (K) and diffuses throughout the body in the same way as K 3). Furthermore, Cs+ inbacteria is taken up by the cells via the K+ uptake system. On the other hand, as there is noCs+ excretion system and Cs+ accumulates in the cells, Cs+ is toxic to bacteria 4). Meanwhile,in our laboratory, Microbacterium sp. TS-1, which is resistant to high cesium concentrations,has been isolated from Jumping Spiders ground extract 5). Strain TS1 possesses a Cs+/H+
antiporter (CshA) capable of taking H+ into cells and exporting Cs+. This study aims to utilize
CshA to construct a system for recovering Cs from pollutants.
Methods
Developing a simple membrane vesicle recovery system for practical applications is necessary
as a technology for using inverted membrane vesicles containing the abovementioned CshA
enzyme to recover and concentrate large amounts of Cs+ from the environment. Practicalapplications cannot be realized unless a more efficient membrane vesicle collection system
becomes available. In addition, an ultracentrifuge is required to collect the inverted membrane,
which is practically unsuitable. Therefore, we attempted to optimize the recovery conditions.
Escherichia coli inverted membrane vesicles expressing CshA were confined to a sphericalgel with a diameter of approximately 2 mm using calcium alginate. The requirements forcreating a spherical gel of calcium alginate
embedded with membrane vesicles
expressing CshA were determined by trial
and error, and the optimal conditions forthe mixing ratio were thereby determined.
In this experiment, we used the highly Csresistant E. coli strain ZX1 obtained bytransforming E. coli Mach1 with CshA
possessed by the TS-1 strain. In addition,
an E. coli KNabc strain in which the major
Na+/H+ antiporter was deleted was used asa negative control. First, high-pressure cell
disruption was performed to prepare inverted membrane vesicles from the ZX-1 strain usinga French press (Glen Mills). The prepared inverted membrane vesicles were wrapped in 1%
sodium alginate aqueous solution using a double-tube nozzle and dropped into 100 mM
calcium chloride aqueous solution to form a spherical gel (Figure 1). Twenty spherical gelswere aliquoted into 15 ml centrifuge tubes, and 3 ml each tube was dispensed into a 30 mM
Tris-HCl buffer (pH 8.5) mixture containing 30 mM CsCl and 2.5 mM succinic acid. The
mixture was shaken for 1 h, and the supernatant was collected. The Cs+ concentration in thesupernatant was measured using a flame photometer (BWB Technologies).
Results
The everted membrane
vesicles from strains
KNabc and ZX1 wereprepared withconcentrations of 10mg/ml, 20 mg/ml, and40 mg/ml, respectively,and alginate gels without
everted membrane
vesicles were mixed
with 30 mM CsCl and2.5 mM succinate. Thevesicles were thenincubated in 30 mM Tris-HCl buffer (pH 8.5) containing acid for 1 h with shaking. Adecrease in Cs+ concentration was confirmed in both the KNabc and ZX1 strains (Figure 2).

In particular, the everted membrane vesicles of ZX1 recovered more Cs+ than those ofKNabc. In addition, the Cs+ concentration in the gel alone, without the addition of everted
membrane vesicles, was consistent with the decrease in Cs+ concentration accompanied byan increase in volume when 20 gels were added. Therefore, we concluded that Cs+ is notdirectly adsorbed onto the gel.
Conclusion
We previously confirmed that Cs+ can be recovered using an everted membrane expressing
CshA. However, when using the spherical gel prepared by mixing everted membrane vesicles
with alginic acid, selective recovery of Cs+ by CshA was not observed. Therefore, in this study,
we attempted to recover Cs+ using an alginate spherical gel embedded with everted membranevesicles expressing CshA using a double-tube nozzle (Figure 1). In the alginate spherical gelembedded with everted membrane vesicles, the Cs+ recovery rate increased as theconcentration of the embedded everted membrane vesicles increased. Consequently, we havedemonstrated that Cs+ can be recovered in the form of a spherical gel with an approximate
diameter of 2 mm, eliminating the need to retrieve the everted membrane vesicles using anultracentrifuge. We believe increasing the number of everted membranes embedded in thealginate gel can further increase Cs+ recovery. Various conditions are currently being
considered to further improve the Cs+ recovery rate.
References
1) TEPCO treated water portal site.
https://www.tepco.co.jp/en/decommission/progress/watertreatment/alpsstate/index-e.html

2) Lopez-Fernandez, M., Jroundi, F., Ruiz-Fresneda, M., and Merroun, M. (2021) Microbialinteraction with and tolerance of radionuclides: underlying mechanisms and biotechnologicalapplications. Microb. Biotechnol. 14: 810-828.
3) Melnikov P. and Zanoni L.Z. (2009) Clinical effects of cesium intake. Biol. Trace Elem.
Res. 135: 1-9.
4) Avery, S.V. (1995) Caesium accumulation by microorganisms: uptake mechanisms, cationcompetition, compartmentalization, and toxicity. J. Ind. Microbiol. 14: 76-84.
5) Koretsune, T., Ishida, Y., Kaneda, Y., Ishiuchi, E., Teshima, M., Marubashi, N., Satoh, K.,and Ito, M. (2022) Novel cesium resistance mechanism of alkaliphilic bacterium isolated fromjumping spider ground extract. Front. Microbiol. 13: Article number 841821.