Characterization of Cesium Uptake Mediated by a Potassium Transport System of Bacteria in a Soil Conditioner

Abstract

We found that bacteria in a commercial soil conditioner sold in Ishinomaki, Miyagi, exhibited concentrative and saturable cesium ion (Cs⁺) uptake in the natural range of pH and temperature. The concentration of intracellular Cs⁺ could be condensed at least a few times higher compared with the outside medium of the cells. This uptake appeared to be mediated by a K⁺ transport system, since Cs⁺ uptake was dose-dependently inhibited by potassium ion (K⁺). Eadie-Hofstee plot analysis indicated that the Cs⁺ uptake involved a single saturable process. The maximum uptake amount (J_max) was the same in the presence and absence of K⁺, suggesting that Cs⁺ and K⁺ uptakes were competitive with respect to each other. These bacteria might be useful for bioremediation of cesium-contaminated soil.

Cesium-137 (¹³⁷Cs, half-time=30.17 years) was one of the main radioactive materials released in the Fukushima nuclear power plant accident after the tsunami in March, 2011. The released ¹³⁷Cs entered the soil from wastewater or rain, presenting a serious risk of contamination of agricultural products and the environment. One approach to reduce or eliminate environmental hazards resulting from uptake of toxic metals, chemicals and other hazardous wastes is bioremediation using living microorganisms.¹⁾ Indeed, several bacteria are able to accumulate cesium ion (Cs⁺).^2–4) Also, Cs⁺ uptake by bacteria is inhibited by potassium ion (K⁺).^2,5) Thus, Cs⁺ might be accumulated via K⁺ transport system(s). All bacteria contain multiple K⁺ uptake systems on the cell membrane. For example, the properties of three K⁺ transport systems (Kdp, Trk and Kup) in Escherichia coli (E. coli) have been reported.^6,7) Among them, the Kdp system transported K⁺ with high affinity, but did not transport Cs⁺.⁸⁾ The Trk system transported K⁺ and rubidium, but not Cs⁺.⁶⁾ On the other hand, the Kup (TrkD) system was found to transport Cs⁺ with moderate rate and affinity.⁶⁾ Furthermore, other bacteria such as Rhodococcus erythropolis CS 98 and Rhodococcus sp. Strain CS402 were suggested to accumulate Cs⁺ via a K⁺ transport system that was different from the Kup system of E. coli.⁵⁾ Such bacteria might be useful for bioremediation of radioactive cesium-contaminated soils.

We found that bacteria in a soil conditioner sold in Ishinomaki, Miyagi, situated near the area contaminated during the nuclear power plant accident, exhibited Cs⁺ uptake. Here, we investigated the mechanism and kinetics of Cs⁺ uptake by these bacteria.

MATERIALS AND METHODS

Materials

Soil conditioner containing microorganisms (1.8×10⁸ cells/1 g) was supplied by Fukujyu Corporation (Ishinomaki, Miyagi, Japan). The composition of microorganisms was 50% Lactobacillus, 12.5% Bacillus, 12.5% Actinomycetes, 12.5% Chlorella and 12.5% VS34 fungus. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) and N-cyclohexyl-2-aminoethanesulfonic acid (CHES) were purchased from Dojindo Laboratories (Kumamoto, Japan). Fluorescein isothiocyanated-labeled dextran (FITC-dextran) was purchased from Sigma-Aldrich (Tokyo, Japan). Bio-Rad DC Protein Assay Kit was purchased from Bio-Rad Laboratories (Nippon Bio-Rad Laboratories). Other materials were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Isolation of Cells

Soil conditioner (2 g dry weight) was suspended in 50 mL of 200 mM HEPES buffer (pH 7.5). Cells were purified by filtration through a 4 µm pore size filter paper (5B quantitative ash-less filter paper, ADVANTEC (Tokyo Japan)). Subsequently, K⁺ in cells was depleted by a 30 min treatment with 10 mM 2,4-dinitrophenol (DNP) in HEPES buffer at 25±1°C, as reported elsewhere.^6,7,9) Most K⁺ in the cells is removed by this treatment and over 90% is lost within the first few minutes.⁷⁾ Cells were collected by centrifugation at 6000×g for 30 min and washed once with HEPES buffer. The concentrations of cells were determined by measurement of the absorbance (optical density) at 600 nm in all experiments.

Cs⁺ Uptake Experiments

Cells (6.00×10⁷ cell/mL) were cultured in 10 µM CsCl in HEPES–glucose buffer (pH 7.5) containing 200 mM HEPES with 10 mM glucose^6,10) for designated periods at 25±1°C with shaking (TAITEC, 160 cycles/min). In the pH dependence experiment, cells were suspended in the following buffers (200 mM) containing 10 µM CsCl and 10 mM glucose: PIPES for pH 6.5 and 7.0, HEPES for pH 7.0, 7.5 and 8.0, TAPS for pH 8.0, 8.5 and 9.0, CHES for pH 9.0 and 10.0. The final pH was adjusted with 5 M NaOH. Cells were cultured in 10 µM CsCl in HEPES–glucose buffer with shaking to study the effect of temperature (4°C, 25°C or 37°C). Cells were cultured in 10 µM CsCl HEPES–glucose buffer containing 1 or 3 mM KCl to study the inhibitory effect of K⁺ on Cs⁺ uptake. The concentration dependence of Cs⁺ uptake was examined at 25±1°C with DNP-treated cells suspended in HEPES–glucose buffer containing 0 µM to 10 mM CsCl with or without 3 mM KCl. After uptake, cells were harvested by centrifugation (9000×g, 10 min) through 0.45-µm pore size membrane filters (MF-membrane filter, Millipore). The Cs⁺ contents of cell pellets (50 µL) were measured by the graphite furnace method using a High-Resolution Continuum Source Atomic Absorption Spectrometer (ContrAA 700 Analytik Jena, Germany).

Estimation of Cell Volume and Amount of Protein

Soil conditioner (3 g dry weight) was suspended in 50 mL of 200 mM HEPES buffer (pH 7.5). Cells were purified by filtration through 4 µm pore size filter paper and collected by centrifugation at 6000×g for 30 min. The supernatant was discarded and cells were suspended in 100 µL of 200 mM HEPES buffer containing glucose (10 mM), antipyrine (200 µM: C_A0) and FITC-dextran (100 µM: C_F0).^11–13) After 30 min of incubation at 25±1°C, cells were filtered through 0.45-µm pore size membrane filters. Antipyrine concentration in the filtrate (C_A) was measured by high-performance liquid chromatography (HPLC) using a constant-flow pump (Shimadzu LC-10AT), a UV detector (Shimadzu SPD-10A), an automatic sample injector (Shimadzu SIL-10A) and an integrator (Shimadzu C-R7APlus), with detection at 254 nm. The mobile phase, which was delivered at a flow rate of 1 mL/min, consisted of acetonitrile and 10 mM KH₂PO₄ (pH 4.68) (15 : 85, v/v). Concentration of FITC-dextran in the filtrate (C_F) was measured using a Wallac 1420 Multilabel Counter (PerkinElmer, Inc., Waltham, Massachusetts). Cell concentration (C_cell) was measured in terms of absorbance (optical density at 600 nm). Cell volume (V_cell) was calculated using the following formula:

  

The protein concentration was determined using Bio-Rad DC protein Assay Kit by the Lowry method with bovine serum albumin as a standard.¹⁴⁾ The protein content per 10⁶ cells was calculated.

Data Analysis

The kinetic parameters for the uptake of Cs⁺ by cells were evaluated according to the following equation.

  

J_max is the maximum uptake amount (pmol/4 h/10⁶ cells) via the carrier-mediated process, S is the concentration of the substrate (µM), K_t is the concentration giving half-saturation (µM) and k_d is the first-order rate constant (µL/4 h/10⁶ cells). All experiments were carried out in triplicate. Arithmetic means and standard errors were calculated with Studentʼs t-test statistics using Excel for Windows software. Differences among three groups were analyzed using the ANOVA test. A difference between the means was considered to be significant when p was less than 0.05.

RESULTS

Time Dependence of Cs⁺ Uptake

Figure 1 showed the time course of Cs⁺ uptake into cells. The number of cells did not change markedly during the incubation period (2.4–3.6×10⁶ cells/50 µL). Cs⁺ uptake was linear from 3 to 8 h, and the initial rate was 0.38 pmol/h/10⁶ cells. Therefore, a 4-h period was selected for further experiments. The Cs⁺ concentration in cells was 3.52±0.33 pmol/10⁶ cells after 4-h incubation and 5.46±0.96 pmol/10⁶ cells after 24-h incubation. Based on a cell volume of 1.64±0.65×10⁻¹³ L, the calculated intracellular Cs⁺ concentrations after 4 and 24-h incubation were 21.52±3.45 µmol/L and 33.31±10.01 µmol/L, respectively. Therefore, the intracellular Cs⁺ concentration was increased 2.15 times after 4-h incubation and 3.33 times after 24-h incubation, compared with that of the medium.

Fig. 1. Time Dependence of Cs⁺ Uptake

Cells were incubated in the presence of 10 µM CsCl in HEPES–glucose buffer for 24 h. Each value of intracellular Cs⁺ concentration represents the mean±S.E. of three replicate experiments.

pH Dependence on Cs⁺ Uptake

Cs⁺ uptake was dependent on external pH (Fig. 2), but was not dependent on the nature of the buffers used. The uptake was highest at pH 7.0 to 8.0, and greatly decreased above pH 8.5. More than 60% of the Cs⁺ uptake activity at pH from 7.0 to 8.0 was retained over the pH range of 6.5 to 8.5.

Fig. 2. pH Dependence of Cs⁺ Uptake

Cells were suspended in different buffers (200 mM) containing 10 µM CsCl (see text) adjusted to the desired pH values. Symbols: ○, PIPES; ●, HEPES; △, TAPS; ▲, CHES. Cs⁺ uptake was measured after 4-h incubation. Each value represents the mean±S.E. of three replicate experiments.

Temperature Dependence on Cs⁺ Uptake

Cs⁺ uptake showed an increasing trend as the temperature increased (Fig. 3). Uptake at 4°C was lowest and that at 37°C was highest. However, the differences were not statistically significant.

Fig. 3. Temperature Dependence of Cs⁺ Uptake

Cells were incubated in presence of 10 µM CsCl in HEPES–glucose buffer at 4, 25 and 37°C. Cs⁺ uptake was evaluated after 4-h incubation. Each value of intracellular Cs⁺ concentration represents the mean±S.E. of three replicate experiments.

Inhibitory Effect of K⁺ on Cs⁺ Uptake

Cs⁺ uptake was dose-dependently inhibited by K⁺ (Fig. 4).

Fig. 4. Inhibitory Effect of K⁺ on Cs⁺ Uptake

The cells were suspended in 10 µM CsCl in HEPES–glucose buffer, pH 7.5, containing 0 mM, 1 mM or 3 mM KCl. Each value of intracellular Cs⁺ concentration represents the mean±S.E. of three replicate experiments. The significance of differences between 0 mM and 3 mM KCl was determined by means of the t-test. * p<0.05.

Concentration Dependence of Cs⁺ Uptake

As shown in Fig. 5, Cs⁺ uptake increased with increasing Cs⁺ concentration in the medium, reaching a plateau at 1.0 mM. An Eadie–Hofstee plot (Fig. 5, outer) indicated that a single saturable process was involved. The value of J_max was not altered in the presence of 3 mM K⁺. Kinetic analysis gave J_max, K_t, k_d and k_i (inhibition constant of K⁺) values of 556 pmol/4 h/10⁶ cells, 256 µM, 0.0188 µL/4 h/10⁶cells and 2.2 mM, respectively. Protein amount was 0.373 µg/10⁶ cells. If this is assumed to be equal to the dry cell weight, Jmax and J_max/K_t can be calculated as 9.65 µmol/min/g dry cell and 0.038 L/min/g dry cell, respectively.

Fig. 5. Concentration Dependence of Cs⁺ Uptake

The concentrations of CsCl (S) used were 0 µM, 10 µM, 30 µM, 100 µM, 300 µM, 1 mM, 3 mM and 10 mM, with or without 3 mM KCl. Symbols: ○, CsCl without KCl; ●, CsCl with 3 mM KCl. Cs⁺ uptake (J) was measured after 4-h incubation. The result of Eadie–Hofstee plot analysis is also shown. Each value represents the mean±S.E. of three replicate experiments. If the S.E. is not shown, it is smaller than the symbol.

DISCUSSION

Information on Cs⁺ uptake by bacteria is limited.^4,15) Here, we focused on the mechanism and kinetics of the Cs⁺ uptake of bacteria found in soil conditioner sold in Ishinomaki, Miyagi. The Cs⁺ uptake was saturated within 24 h. Moreover, the intracellular concentration of Cs⁺ was increased several-fold over that of the medium. The most common range of soil pH is 4 to 8, and the range for optimal availability of plant nutrients for most crops is pH 6.5 to 7.0.¹⁶⁾ The optimal pH of Cs⁺ uptake was from pH 7.0 to 8.0 (Fig. 2), in agreement with previous findings.⁴⁾ More than 60% of the Cs⁺ maximum uptake activity was retained over the pH range from 6.5 to 8.5. Moreover, the Cs⁺ uptake was not significantly affected by temperature over the range of 4 to 37°C, so seasonal variation in temperature may not have a marked effect. Therefore Cs⁺ uptake is likely to take place in soil under natural conditions.

It is thought that cellular Cs⁺ uptake is mediated by K⁺ transport systems, as in Rhodococcus,^5,17) E. coli ⁶⁾ and Chlorella salina,³⁾ since Cs⁺ uptake was dose-dependently inhibited by the presence of K⁺. Eadie–Hofstee plot analysis confirmed that the Cs⁺ uptake involved a single saturable process. Furthermore, because Jmax was the same in the presence and absence of 3 mM K⁺, the Cs⁺ and K⁺ uptakes appear to be competitive with respect to each other.

The E. coli Kdp and Trk systems do not transport Cs⁺, so the K⁺ transport system of the bacteria may be different from these systems. On the other hand, the K_t, J_max and J_max/K_t values that we obtained (Table 1) were similar to those of Rhodococcus erythropolis CS98 or Rhodococcus sp. Strain CS402.^5,17) Rhodococcus belongs to Actinobacteria, which also includes Actinomycetes species present in the soil conditioner. Thus, Actinomycetes might be a major contributor to Cs⁺ accumulation. Further work is needed to identify bacteria in the soil conditioner that are involved in Cs⁺ uptake.

Table 1. Kinetic Parameters of Cs⁺ Transport by Bacteria

Strain or system	Parameter			Reference(s)
	Kt (µM)	Jmax (µmol/min/g dry cell)	Jmax/Kt (L/min/g dry cell)
Rhodococcus erythropolis CS98	136	16	0.118	5
Rhodococcus sp. Strain CS402	434	25	0.058	5
Rhodopseudomonas capsulatus	3000	2	0.00067	5, 17
E. coli Kup	5000	17	0.0034	5, 6
Chlorella salina	500	33.3 nmol/h/106 cells	66 µL/h/106 cells	3
Bacteria in this paper	256	9.65 (556 pmol/4 h/106cells)	0.038 (0.543 µL/h/106 cells)	This work

E. coli Kdp and E. coli Trk systems do not take up Cs⁺.^5,6)

In conclusion, we found that bacteria in the soil conditioner rapidly accumulated Cs⁺ up to saturation levels under natural conditions of pH and temperature. Moreover, the efflux capability of the K⁺ channel for Cs⁺ is only 2–20% of that for K⁺,^18,19) so it is plausible that intracellular accumulation of Cs⁺ could be substantial and prolonged. Preliminary results of field studies using ¹³⁷Cs-contaminated soils indicate that this approach may be effective for removing ¹³⁷Cs from soil.

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