Strains
Two commercial shiitake strains were purchased, which are referred to as Strain 1 and Strain 2, respectively, hereafter in consideration of reputational damage. Strain 1 and Strain 2 are mainly used for sawdust medium cultivation and bed-log cultivation, respectively.
Bed-log cultivation for estimating the sampling position of bed log for accurate TF measurement
Konara oak bed logs that were approximately 11 cm in diameter and 90 cm long and had been inoculated with Strain 2 approximately 2 years previously were purchased from a grower. A hole with a diameter of 1 cm was made in the wood (depth = 3.2 cm) in the longitudinal center of each bed log, and antibiotic assay filter paper (49005010; ADVANTEC Co., Ltd., Tokyo, Japan) impregnated with a CsCl solution containing 1.0 mg 133Cs was placed in the bottom of the hole. The hole was then sealed with Japanese beech (Fagus crenata) sawdust. Three bed logs were prepared for the experiment. The bed logs were soaked in tap water for approximately 16 h, and maintained at 15 °C and a relative humidity of ≥ 90% under fluorescent lamp irradiation for fruiting body development. Each fruiting body was harvested when the cap was approximately 80% open, and the point on the bed log where the fruiting body developed was recorded. Because the stipes of shiitake cultivated in bed-log cultivation are cut off before the ingredients are analyzed [13], the stipe was discarded and only the cap was sliced, dried at 105 °C, and milled with a Millser (IFM-620DG; Iwatani Co., Tokyo, Japan). The concentration of 133Cs in each cap was determined by subtracting 0.59 mg/kg from the measured value, which equated to the cap 133Cs concentration in an untreated bed log. The cap 133Cs concentrations at the sampling locations are shown as a contour graph using SigmaPlot version 14.0 (Systat Software, Inc., California, USA), with the circumferential direction of the bed log aligned along the x-axis, the axial direction of the bed log aligned along the y-axis, and the perforated portion is represented by (0, 0).
Bed-log cultivation for estimating the 137Cs TF and the 133Cs TF
The same kind of konara oak bed logs were purchased from the same grower as described above, and the fruiting bodies were developed and harvested, the point on the bed log where the fruiting body developed was recorded, and the caps were treated in the same manner as described above. Once fruiting body development was completed, each bed log was cut radially 3 cm axially from the recorded point, and the resulting disc was then cut axially along the line connecting the center of the bed log and the place 5 cm circumferentially away from the recorded point (Fig. 1). The bark and wood were separated from the resulting wedge-shaped blocks and dried at 105 °C. The wood was then crushed with a cutting mill (P-15; Fritsch Japan Co., Ltd., Kanagawa, Japan) and both the bark and wood were milled separately with the Millser.
Sawdust medium cultivation for estimating the effect of medium 133Cs concentrations on the cap 133Cs concentrations
Commercially available Japanese beech sawdust and rice bran were used as the sawdust medium. Sawdust and rice bran were mixed at a ratio of 75:25 (w/w), and the sawdust medium 133Cs concentration was adjusted to 5 levels [control, 0.50, 0.10, 0.15, and 0.20 mg/kg (w/w)] using CsCl. After adjusting the water content to 65% (w/w), a 1 kg mixture was packed into a plastic bag and autoclaved at 121 °C for 90 min. Three sawdust media were prepared for each CsCl concentration. The sterilized sawdust media were inoculated with the mycelia of the both strains that had been cultivated for 2 weeks in 40 mL SMY liquid medium [10 g/L sucrose, 10 g/L malt extracts (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan), and 4 g/L yeast extracts (Oxoid Ltd., Hampshire, England)] and cultured at 22 °C and 70% relative humidity in the dark for 16 weeks [14]. The fruiting bodies that developed were then treated in the same manner as described above for bed-log cultivation. Once fruiting body development was completed, the sawdust medium was dried at 105 °C, crushed with a pulverizer (V-360; HORAI Co., Ltd., Osaka, Japan), and milled with the Millser.
Sawdust medium cultivation for estimating the effect of the nutrient concentration on TF
Japanese beech sawdust and rice bran were mixed at 95:5, 90:10, 85:15, 80:20, 75:25, and 70:30 (w/w), and the 133Cs concentrations in these 6 media were adjusted to 0.20 mg/kg (w/w) using CsCl. The cultivation was performed in the same manner as described above for sawdust medium cultivation.
Sawdust medium cultivation for estimating the effect of the cultivation period on TF
Japanese beech sawdust and rice bran were mixed at a ratio of 95:5 (w/w), and the 133Cs concentrations in the resulting sawdust media were adjusted to 0.20 mg/kg (w/w) using CsCl. The cultivation was performed in the same manner as described above for sawdust medium cultivation, but cultivation periods were set for 5 levels (16, 20, 24, 28, and 32 weeks).
Sawdust medium cultivation for estimating the differences between the 137Cs TF and 133Cs TF, and the differences between strains
Radioactive contaminated sawdust and rice bran were mixed at a ratio of 95:5 (w/w). The radioactive sawdust was collected from a contaminated site in Fukushima prefecture in July 2011 and consisted of mainly hardwood (unknown species). The cultivation was performed in the same manner as described above for sawdust medium cultivation, but the cultivation period was 28 weeks.
Elemental analysis
The total 137Cs (to137Cs) concentrations in the bark and wood samples were measured by filling 20-mL vials (6000477; PerkinElmer Japan Co., Ltd., Kanagawa, Japan) with each sample and measuring the to137Cs concentration twice per sample for 1 h each using a gamma counter (2480 WIZARD2; PerkinElmer Japan Co., Ltd.). The to137Cs concentration in the sawdust medium was measured by placing each sample in a 0.7-L Marinelli beaker or U-8 container, depending on the sample volume, and using a high-purity germanium detector (GEM20-70; SEIKO EG&G Co., Ltd., Tokyo, Japan). The cap 137Cs concentrations in the bed-log cultivation were measured by mixing each sample cap with a cap of known 137Cs concentration (6.3 Bq/kg) to give a total amount of approximately 9 g, because each sample volume was too small to measure with the gamma counter. The 137Cs concentration in the resulting mixture was then measured in the same manner as described above using the gamma counter and the cap 137Cs concentration was calculated from its weight ratio in the mixed sample. The cap 137Cs concentrations in the sawdust medium cultivation were measured by enclosing each sample in a U-8 container and using the high-purity germanium detector, as described above.
To determine the ex137Cs concentration in the sawdust medium, 10 mL of 1 mol/L ammonium acetate solution adjusted to pH 7.0 with acetic acid or ammonia solution was added to approximately 1 g of each sawdust sample. The mixture was then swirled for 16 h with a rotating incubator (RT-50; TAITEC Corp., Saitama, Japan) and centrifuged at 5000 × g for 10 min. The resulting supernatant was filtered through a 0.50-μm hydrophilic polytetrafluoroethylene (PTFE) membrane filter, and the ex137Cs concentration was measured three times per sample for 2 h each using the gamma counter, as described above.
To measure the total 133Cs (to133Cs) concentration in the all samples, 14 mL of concentrated nitric acid (Ultrapure-100; KANTO CHEMICAL Co., Inc., Tokyo, Japan) was added to approximately 0.5 g of sample in the lower container of an ECO-PRE (OD-98-100; ACTAC Co., Kanagawa, Japan), and 6 mL of 5% nitric acid was added to the trap part. Digestion was then carried out by repeating nine cycles of 210 °C for 20 min and 100 °C for 25 min. The resulting solution was filtered through the same PTFE membrane filter, and the to133Cs concentration was measured using an inductively coupled plasma mass spectrometer (ICP-MS) (7700x; Agilent Technologies International Japan Ltd., Tokyo, Japan). The cap cultivated in the bed-log cultivation was mixed with supplemental sample for measuring the cap 137Cs concentration, and the supplemental cap 133Cs concentration was 0.32 mg/kg, so the cap 133Cs concentration in the bed-log cultivation was calculated as described above.
To measure the exchangeable 133Cs (ex133Cs) concentration in the sawdust medium, the ex133Cs was extracted as described above for ex137Cs, and measured as described above using ICP-MS.
The nitrogen (N) concentration in the sawdust medium was measured with an NC analyzer (Sumigraph NC-22F; Sumica Chemical Analysis Service Ltd., Osaka, Japan) using approximately 20 mg sample.
Calculation
The sawdust medium Cs concentrations were changed depending on the cultivation stages, so the TFs based on the medium Cs concentration at the start of cultivation (TFSC), before fruiting body development (TFBD), and after fruiting body development (TFAD) were calculated as follows:
$${\text{TF}}_{{{\text{SC}}}} = \frac{{\text{Fruiting body Cs concentration}}}{{\text{Medium CS concentration at the start of cultivation}}},$$
(1)
$$\begin{gathered} {\text{TF}}_{{{\text{BD}}}} = \frac{{\text{Fruiting body Cs concentration}}}{{\text{Medium Cs concentration before fruiting body devrlopment}}} \\ = \frac{{\text{Fruiting body Cs concentration}}}{{{ }\frac{{{\text{Medium Cs amount after fruiting body development }} + {\text{fruiting body Cs amount}}}}{{{\text{Medium weight after fruiting body development }} + {\text{ fruiting body weight}}}}}}, \\ \end{gathered}$$
(2)
$${\text{TF}}_{{{\text{AD}}}} = \frac{{\text{Fruiting body Cs concentration}}}{{\text{Medium Cs concentration after fruiting body development}}}.$$
(3)
Since there were two kinds of Cs concentration (toCs concentration and exCs concentration) and two kinds of Cs (137Cs and 133Cs), they were reported as to137Cs TF, to133Cs TF, ex137Cs TF, and ex133Cs TF, respectively. The amount of fixed Cs (fxCs) was calculated as follows:
$${\text{Fixed Cs amount}} = {\text{total Cs amount}} - {\text{exchangeable Cs amount}}{.}$$
(4)
The exCs solubilization ratio was calculated as follows:
$${\text{exCs Solubilization ratio}} = \frac{{{\text{fxCS}}_{{{\text{SC}}}} - {\text{fxCs}}_{{{\text{AD}}}} }}{{{\text{fxCs}}_{{{\text{SC}}}} }},$$
(5)
where fxCsSC and fxCsAD were the fxCs amounts at the start of cultivation and after harvesting fruiting body, respectively. The wet weight-based TF was calculated as follows:
$${\text{Wet weight}} - {\text{based TF}} = \frac{{\frac{{\text{Fruiting body Cs concentration}}}{{1 - \frac{{{\text{Fruiting body water content }}\left( {\text{\% }} \right)}}{100}}}}}{{\frac{{\text{Medium Cs concentration}}}{1 - 0.12}}},$$
(6)
because the water content of the culture medium is specified as 12% [15]. The significance level was set to 0.05.