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Site informationWe collected radioactivity data of wild mushrooms and wild edible plants from inspection results of specimens brought in by residents in Kawauchi Village, which is located 12–30 km away from the FDNPP (Fig. 1). Kawauchi Village is considered small, with an area of 197.4 km2, and a population of about 2500 (2820 in 2010 and 2518 in 2021)48. It is located in the middle of the Abukuma Highlands, where the elevation ranges from 270 to 1,192 m above the sea level. It has a forest coverage of 89.0%, which is higher than the average for Fukushima Prefecture (71%) and Japan as a whole (69%)49. 137Cs deposition in the village ranged from 42 to 960 kBq/m2 in 2011, estimated from an aircraft monitoring28. Before the accident, its residents were accustomed to gathering wild foods, such as wild edible mushrooms, plants, mammals, and wild honey50; many have been brought in for inspection. Information on collection areas of sub-village levels, called “Ko-aza” in Japanese, is also recorded. For these reasons, we thought that the data of the brought in inspection in Kawauchi Village would possess high value as data for inter-species and inter-region analysis on the wild mushrooms and edible plants’ radioactivity concentrations.Radioactivity data of mushrooms and wild plantsFukushima Prefecture sets up a system for each municipality to inspect radioactivity in vegetables and mushrooms consumed by residents, and Kawauchi Village started its inspection program in May 2012. Simple inspection machines are set up at public facilities, and inspections are conducted upon application by residents. In Kawauchi Village, the location of samples inspected was requested at the sub-village level. The inspection results were regularly reported in the village newsletter, along with the inspection date, inspected food, and collection location. The data compiled from May 2012 to March 2020 was provided to us through the village officials. Orita et al. analyzed the same inspection data of agricultural products in Kawauchi Village24. They used 7668 food data from April 2013 to December 2014, including 1986 wild plants and mushrooms data for internal radiation exposure assessment. Some of their data overlap with the data used in our analyses.System of monitoring radioactivity in Kawauchi VillageKawauchi Village started the brought in inspection in May 2012, and there is a maximum of eight inspection stations and currently three stations managed by residents. In the inspection sites, there are four types of NaI (Tl) or CsI (Tl) scintillation detectors. The machine names are Triathler Becquerel Finder (Hidex, Oy, Finland), Captus-3000A (Capintec, NJ), CAN-OSP-NAI (Hitachi Aloka, Tokyo, Japan), and FD-08Cs1000-1 (X-Ray Technology, Osaka, Japan). Table S4 shows the specifications of the machines51,52,53. All instruments have been confirmed to meet the radiocesium screening method requirements for food53. Among these machines, FD-08Cs1000-1 can measure radioactivity non-destructively, and the others conduct destructive measurements. The sample weight is approximately 500 g, and the counting time is 30 min. FD-08Cs1000-1 outputs the summed concentration of the two radiocesium nuclides (134Cs and 137Cs), and its detection limit is 10 Bq/kg (for total 134Cs + 137Cs). Each of the other three machines separately outputs the concentrations of 134Cs and 137Cs, and the detection limit is 10 Bq/kg for each radionuclide. Energy calibrations and background checks were performed daily, and the accuracy was periodically verified with brown rice whose radiocesium concentration was verified by calibrated high-purity Germanium (HPGe) detectors installed in the Fukushima Nuclear Center49. Table S4 shows the results of quality control using brown rice.Data preparation of radioactivity of samplesFrom the radioactivity data of wild mushrooms and plants, we picked up data that met the following criteria;

Data have information of sampling location at sub-village levels

Items that are not confirmed to be cooked products such as “boiled” or “dried.”

Species with more than ten samples in which radiocesium was detected.

In cases where mushrooms and wild plants were given in dialects, we confirmed the species’ names with residents. The names of the species were determined from the Japanese names of the items, but in some cases, it was not possible to distinguish between Cortinarius salor (“Murasakiaburashimejimodoki” in Japanese) and C. iodes (“Murasakiaburashimeji”), considered to be closely related species, so the two were mixed for analysis. The leaf stalk and scape of Petasites japonicus (Japanese butterbur) are called “Fuki” and “Fukinotou” in Japanese, respectively, and are registered separately. Therefore, despite being the same species, they were distinguished in the analysis. In this data, there were not sampling date but measurement date. Therefore, the date of measurement and sample collection were assumed to be the same.The 137Cs concentration results were used in the model analysis. The reason for not using the134Cs concentration among the measured values is explained in the subsection of “Bayesian estimation”. 137Cs concentrations were decay-corrected to March 11th, 2011 for comparison with Komatsu et al. (2019). Based on the assumption that the 134Cs/137Cs ratio at the time of the accident was one54, the summed concentration of 134Cs and 137Cs concentration taken by FD08-Cs1000-1 was converted to a 137Cs concentration, which was decay-corrected to March 11th, 2011, using the following equation;$${}^{137}C{s}_{2011/03/11}=tC{s}_{mathrm{sampling}_mathrm{day}}*frac{{0.5}^{dy/30.17}}{{0.5}^{dy/2.065}+{0.5}^{dy/30.17}}$$In this equation, dy indicates the period from March 11th, 2011, to the date of measuring, and it is expressed by decimal years.Sub-village (“Ko-aza”) boundary map of Kawauchi VillageKawauchi Village comprises eight administrative communities (called “Oh-aza” in Japanese), which are further subdivided into small administrative units known as “Ko-aza”. Here, we refer to these small administrative units as sub-villages. We obtained a sub-village map from the administrative office. The printed map was originally drawn by hand and had been used for village administration. To create a polygon shapefile of the map, we digitized it by scanning, geo-rectifying, and digitizing using GIS software in TNTmips v2014 (MicroImages, Inc, NE) and ArcGIS 10.3 (Esri, Inc, CA). We used this map to associate land names with monthly radioactivity data from samples and to estimate sample collection locations.Deposition dataFor the 137Cs deposition data of this area, we used 250 m grid deposition data measured by the Ministry of Education, Culture, Sports, Science and Technology28,55 and then corrected by Kato and Onda26. We computed the geometric mean value of 137Cs deposition within each sub-village polygon. The 137Cs deposition is also decay-corrected to March 11th, 2011.Bayesian estimationWe constructed a Bayesian model partially modified from Komatsu et al.22 to estimate 137Cs concentration (137Cssample). The model is based on the Gonze and Calmon’s concept of normalized concentration (NC) as expressed by:$$NC= frac{Cs}{D}$$where D indicates the radiocesium deposition amount based on the aircraft monitoring. Then the above equation is transformed and logarithmized to yield;$$mathrm{log}Cs=mathrm{log}NC+mathrm{log}D$$In this expression of the model equation, we further assumed that the logartihm of NC encompassed the summed effects of species identity, collection date, and collection site, and that the logarithm of NC was normally distributed around the estimated mean as per the following equations;$$begin{array}{l}{text{log}}_{10}{hspace{0.17em}}^{137}C{s}_{mathrm{sample}} sim Normal({mu }_{mathrm{sample}},sigma )\ {mu }_{mathrm{sample}} ={text{log}}_{10}N{C}_{mathrm{sp}}+{lambda }_{mathrm{sp}}Y+{text{log}}_{10}{D}_{mathrm{loc}}+{r}_{mathrm{loc}}\ {text{log}}_{10}N{C}_{mathrm{sp}} sim Normal({mu }_{mathrm{sp}},{sigma }_{mathrm{sp}})\ {lambda }_{mathrm{sp}} sim Normal({mu }_{mathrm{lambda sp}},{sigma }_{mathrm{lambda sp}})\ {r}_{mathrm{loc}} sim Normal(0,{sigma }_{mathrm{loc}})end{array}$$where NCsp, λsp, Dloc and rloc indicate characteristics of concentration of species, temporal trends of species, 137Cs deposition of each sub-village area and effects of sub-village on concentration, respectively. rloc is a parameter with zero mean that represents the deviation of the concentration effect from the expected value based on the deposition (Dloc) value at the point of collection. These parameters except Dloc were obtained from hierarchically sampled from normal distribution with hierarchical parameters (μsp, σsp, μλsp, σλsp, σloc). Additionally, rloc was sampled using the Intrinsic Conditional Auto-Regressive (Intrinsic CAR) model56, which is one of the models considering spatial auto-correlation. For samples whose measured radiocesium concentrations were below the detection limit, radiocesium concentration values were estimated by a censoring distribution in which the detection limit was treated as the upper bound57. This model was defined as the “sub-village model” for this research. This model is similar to model 6 in Komatsu et al.22 but differs in that their previous model takes into account 134Cs values and differences between 134 and 137Cs values. Komatsu et al. evaluated the regional trend in the difference between134Cs and 137Cs concentrations across eastern Japan because 137Cs originating from nuclear bomb tests before the FDNPP accident was detected in wild mushrooms sampled in the northern and southern parts of eastern Japan, which are far from the FDNPP and received less deposition from the accident ( More

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Acrylic and polydimethylsiloxane (PDMS) molds preparationThe incubating device for the porous microplate was designed using a CAD software (Solidworks, Dassault Systèmes) and the exported drawing files were used to laser cut 1/4” and 1/8” acrylic sheet (Universal Laser Systems; Supplementary Fig. S2). After washing the cut acrylic parts with deionized water, they were attached by acrylic (Weld-On) and epoxy (3 M) adhesives that were followed by a curing process for ~18 h. Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) was cast onto the acrylic mold and cured at 80 °C for at least 3 h. The PDMS mold was carefully detached from the acrylic surface by dispensing isopropyl alcohol (VWR) into the area between the PDMS and the acrylic molds (Fig. 2a).Fig. 2: Synthesis and characterization of porous microplate.a Procedure to build a porous microplate using polydimethylsiloxane (PDMS) and acrylic molds. b Image of the microplate with an array of culture wells (wall thickness: 0.9 mm). c Scanning electron microscopy image of nanoporous copolymer HEMA–EDMA.Full size imagePorous microplate preparationSynthesis of copolymer HEMA–EDMA was based on previously described protocols [30, 31] and details are given as follows. Prepolymer solution HEMA − EDMA was prepared by mixing 2-hydroxyethyl methacrylate (HEMA; monomer, 24 wt.%, Sigma-Aldrich), ethylene glycol dimethacrylate (EDMA; crosslinker, 16 wt.%, Sigma-Aldrich), 1-decanol (porogen, 12 wt.%, Sigma-Aldrich), cyclohexanol (porogen, 48 wt.%, Sigma-Aldrich) and 2,2-dimethoxy-2-phenylacetophenone (DMPAP; photoinitiator, 1 wt.%). The solution was stored at room temperature without light exposure until further use. Glass slides (75 × 50 mm2, VWR) were chemically cleaned by sequentially soaking in 1 M hydrochloric acid and 1 M sodium hydroxide for one hour, followed by rinsing with deionized water and air drying. The prepolymer solution was cast onto the PDMS mold and a glass slide was placed on the mold. The solution was then polymerized under ultraviolet light with a wavelength 365 nm for 15 min by using a commercial UV lamp (VWR). The photopolymerized device was detached from the PDMS mold and stored in a jar containing methanol (VWR) until further use (Fig. 2a). The jar was refilled with new methanol twice in order to remove the remaining porogen and uncrosslinked monomers from the hydrogel.Upon each incubation experiment with the porous microplate, each device was decontaminated by replacing the solvent with 70% alcohol (VWR) and storing it for 24 h. They were immersed in a pre-autoclaved jar for two weeks with f/2 medium with omitted silicate, where the jar was refilled once with a new sterile medium to adjust its pH for the algal culture and remove any solvent remaining in the hydrogel. Before inoculating microbial cells, each microplate was taken out from the jar and the media remaining on the top surface was removed by absorbing it with a pre-sterilized wipe to minimize the chance for cross-contamination between wells (Fig. 2b).Scanning electron microscopyPhotopolymerized HEMA − EDMA was removed from methanol and dried in air for at least one week to evaporate the excess solvent. A ~5 × 5 mm2 specimen was collected from the dried copolymer and attached to a pin stub. The stub was loaded on a scanning electron microscope (SEM; MERLIN, Carl Zeiss), and the specimen was characterized with imaging software (SmartSEM, Carl Zeiss) with 16,270X magnification and an operating voltage of 1 kV. The SEM imaging was performed at the Electron Microscopy Facility in the MIT Materials Research Science and Engineering Centers (MRSEC; Fig. 2c).Strains and culturing conditionsAxenic P. tricornutum CCMP 2561 was acquired from the National Center for Marine Algae and Microbiota (NCMA) and shown to be axenic via epifluorescence microscopy and sequencing of the 16 S rRNA gene [11]. P. tricornutum was maintained in f/2 medium with 20 g L−1 commercially available sea salts (Instant Ocean, Blacksburg) and with omitted silicate, which we will refer to as f/2-Si [11, 16]. Batch cultures were grown at 20 °C with a 12 h light/12 h dark diurnal cycle and a light intensity of 200 μmol photons m−2 s−1 (Exlenvce). Every 2–3 weeks, axenic cultures were monitored for bacterial contamination by streaking culture samples on marine broth agar [33], that tests for contamination by bacteria that can grow on agar media and is not definitive. Every 6–12 months, every axenic and bacterial co-culture of P. tricornutum was inspected for the absence/presence of bacteria by staining the cellular DNA with 0.1% v/v SYTO BC Green Fluorescent Acid Stain (Thermofisher, Supplementary Fig. S1).Bacterial community samples (referred to as “phycosphere enrichments”) were obtained from mesocosms of P. tricornutum and maintained as previously described [11, 16]. Briefly, an outdoor P. tricornutum mesocosm sample in natural seawater was collected in Corpus Christi, TX and filtered with 0.6–1 µm pores to remove larger algal cells. The bacterial filtrates were inoculated to an axenic algal culture, maintained in f/2-Si media for ~3 months, and washed with a sterile medium to enrich for phycosphere-associated bacteria. These enriched communities were subsequently co-cultured with P. tricornutum in f/2-Si media for ~4 years prior to the start of the experiments.Two bacterial strains, Marinobacter sp. 3-2 and Algoriphagus sp. ARW1R1, were isolated from the phycosphere enrichment samples (Supplementary Table S1). The isolates were either maintained by growing on marine broth agar plates at 30 °C or by co-culturing with P. tricornutum through inoculation of a single colony into the axenic culture.
P. tricornutum culture in porous microplateThree baseline experiments were designed to study how the alga P. tricornutum interacts with its associated bacteria in the porous microplate (Fig. 1). For experiments assessing the algal growth in the microplate, axenic P. tricornutum was acclimated to a copolymer environment in advance by inoculating a stationary phase-culture to a separate microplate. After acclimation for 4 days, the culture was diluted to ~1 × 106 cells ml−1 and transferred to the experimental microplate. Three replicated microplates were placed in a single transparent covered container (128 × 85 × 10 mm3, VWR) which was filled with ~25 ml f/2-Si medium to keep the microplate hydrated throughout the incubation period of 20 days with an initial culture volume of 75 µl (Fig. 1a). The procedures were conducted under a biosafety cabinet to prevent any biological contamination. The cells were incubated under the same conditions as described above for the batch cultures (temperature, light intensity, diurnal cycle).Growth of P. tricornutum was measured by counting cells using a hemocytometer (Electron Microscopy Sciences) or flow cytometry (described later). Specific growth rates were calculated from the natural log of the cell densities in triplicate sampled during an exponential growth phase (day 3 for the batch culture, day 5 for the porous microplate system; Fig. 3a).Fig. 3: Cultivation of P. tricornutum in the porous microplate.a Schematic of a microplate for algal cultivation. b Growth curve and maximal growth rate (inset) comparing the porous microplate with flask culture. Error bars, standard deviation of triplicates. c Cell abundance at center (n = 3) and surrounding (n = 18) wells after incubation. Asterisks denote statistical differences with following levels (two-tailed t-test): ***P  More

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