El ect ron Mi croscopy Observat i on ofBi omi neral i zat i on wi t hi n Wood Ti ssues ofKurogaki
著者Tazaki  Kazue,  Fukuyama At suko,  Tazaki  Fumi e,Takehara Teruaki ,  Nakamura Kei i chi ,  OkunoMasayuki ,  Hashi da Yumi ko,  Hashi da Shozo
j ournal  orpubl i cat i on t i t l e
Mi neral s
vol ume7
number7
page range123
year2017- 07- 19
URLht t p: //hdl . handl e. net /2297/48400
doi: 10.3390/min7070123
minerals
ArticleElectron Microscopy Observation of Biomineralizationwithin Wood Tissues of Kurogaki
Kazue Tazaki 1,*, Atsuko Fukuyama 2, Fumie Tazaki 3, Teruaki Takehara 4, Keiichi Nakamura 5,Masayuki Okuno 6, Yumiko Hashida 1 and Shozo Hashida 1
1Kahokugata Lake Institute, Na 9-9, Kitachujo, Tsubata, Kahokugun, Ishikawa 929-0342, Japan;yumiko977h@gmail.com (Y.H.); hashida@m2.spacelan.ne.jp (S.H.)2Headquarters for Innovative Society-Academia Cooperation, University of Fukui, 3-9-1 Bunkyo,Fukui 910-8507, Japan; atsukof@u-fukui.ac.jp3Department of Occupational Therapy, Osaka Kawasaki Rehabilitation University, 158 Mizuma,Kaiduka, Osaka 597-0104, Japan; taz23a@sky.plala.or.jp4Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahokugun,Ishikawa 929-0342, Japan; take@kanazawa-med.ac.jp5Yamato Environmental Analysis Co., Ltd., 273 Santanda, Kawakita, Nomi-gun, Ishikawa 923-1253, Japan;nakamura@yamatokankyo.co.jp6Department of Earth Sciences, Faculty of Natural System, Kanazawa University, Kakuma-machi, Kanazawa,Ishikawa 920-1192, Japan; mokuno@staff.kanazawa-u.ac.jp*Correspondence: kazuet@cure.ocn.ne.jp; Tel./Fax: +81-76-223-6977
Received: 9 May 2017; Accepted: 15 July 2017; Published: 19 July 2017
Abstract: Interactions between minerals and microorganisms play a crucial role in living wood tissues.However, living wood tissues have never been studied in the ﬁeld. Fortunately, we found severalkurogaki (black persimmon; Diospyros kaki) trees at Tawara in Kanazawa, Ishikawa, Japan. Here,we report the characterization of kurogaki based on scanning electron microscopy equipped withenergy-dispersive spectroscopy (SEM-EDS) and transmission electron microscopy (TEM), associatedwith inductively coupled plasma-mass spectrometry (ICP-MS) analyses, X-ray ﬂuorescence analyses(XRF) and X-ray powder diffraction (XRD) analyses. This study aims to illustrate the ability ofvarious microorganisms associated with biominerals within wood tissues of kurogaki, as shown bySEM-EDS elemental content maps and TEM images. Kurogaki grows very slowly and has extremelyhard wood, known for its striking black and beige coloration, referred to as a “peacock pattern”.However, the scientiﬁc data for kurogaki are very limited. The black “peacock pattern” of the woodmainly comprises cellulose and high levels of crystal cristobalite. As per the XRD results, the blacktaproot contains mineralized 7 Å clays (kaolinite), cellulose, apatite and cristobalite associated withmany microorganisms. The chemical compositions of the black and beige portions of the blackpersimmon tree were obtained by ICP-MS analyses. Particular elements such as abundant Ca, Mg,K, P, Mn, Ba, S, Cl, Fe, Na, and Al were concentrated in the black region, associated with Pb and Srelements. SEM-EDS semi-qualitative analyses of kurogaki indicated an abundance of P and Ca inmicroorganisms in the black region, associated with Pb, Sr, S, Mn, and Mg elements. On the otherhand, XRF and XRD mineralogical data showed that fresh andesite, weathered andesite, and the soilsaround the roots of kurogaki correlate with biomineralization of the black region in kurogaki roots,showing clay minerals (kaolinite) and cristobalite formation. In conclusion, we describe how thebiominerals in the black region in the cellulose within wood tissues grow chemically and biologicallyin the sap under the conditions associated with the beige portions of the taproot. This can explain whythe crystals produce the “peacock pattern” in the kurogaki formed during the year. We conclude thatkurogaki microbiota are from bacteria in the andesitic weathered soil environment, which producesiliciﬁcation. In other words, the patterned portions of kurogaki consist of siliciﬁed wood.
Minerals 2017, 7, 123; doi:10.3390/min7070123www.mdpi.com/journal/minerals
Minerals 2017, 7, 1232 of 21
Keywords: black persimmon tree kurogaki; peacock pattern; biomineralization; microorganisms;XRD; XRF; SEM-EDS; TEM; ICP-MS; kaolinite; cristobalite; apatite; cellulose; Pb; Sr; andesite
1. Introduction
Patterned kurogaki is currently very rare and difﬁcult to ﬁnd. It is an important material formanufacturing furniture, tea ceremony goods, boxes, and other miscellaneous articles in Japan(Figure 1a,b). Here, we report the characterization of kurogaki at Tawara, Kanazawa, IshikawaPrefecture, Japan, based on radioactivity, mineral analyses, chemical analyses, H2O2 reactions, andbiological observations. No report has yet described the results of electron microscopy observations andchemical analyses of kurogaki, limiting our ability to obtain insights into its nature. We thus studied themineralogy, chemistry, and micro-morphology of kurogaki using a combination of micro-techniques.The objective of this study was to illustrate the association of minerals with variousmicroorganisms that are capable of absorbing these elements from weathered andesite soils. The H2O2reaction produced oxygen bubbles within the black portions of the wood, but the beige portions wereunreactive. Ultraviolet analysis indicated color in the purple part of the spectrum in the black “peacockpattern” portions and the black soils taken from around the roots [1].Kurogaki (black persimmon) grows very slowly, has very hard wood, and is known for a strikingblack and beige coloration, referred to as a “peacock pattern” (Figure 1a,b). Patterned kurogaki occursvery rarely, being only one of every 1000–10,000 persimmon trees found in Japan and China. This treewas previously planted in Kanazawa, Japan, and is of historical and artistic importance in KanazawaCity, Ishikawa, Japan, particularly in the Edo period (1603–1868).In this study, we analyzed the mineralogy, chemistry, micro-morphology, and microstructureof kurogaki wood, considering its association with microorganisms, through a combination ofanalytical data obtained from X-ray diffraction (XRD) and inductively coupled plasma-massspectrometry (ICP-MS), X-ray ﬂuorescence analyses (XRF), scanning electron microscopy equippedwith energy-dispersive spectroscopy (SEM-EDS), and transmission electron microscopy (TEM).We investigated the distribution and location, identiﬁcation and structure, as well as differentiationbetween the black “peacock pattern” and ordinary beige wood portions. We analyzed the chemicalcomposition of black “peacock pattern” portions, the associated microstructure, and elementaldistribution in order to clarify the inﬂuences of environmental soil and water quality on theseproperties. The results provided evidence of the microorganisms’ ability to grow in the black “peacockpattern” portions, immobilizing elements, and performing biomineralization and phytoremediation,in particular, within wood tissues of kurogaki.
2. Materials and Methods
2.1. Exploring Kurogaki Field and Investigated Specimens
Kurogaki samples were obtained from 20 trees from Tawara, Kanazawa, Japan. They were cutdown in December 2016 and subjected to chemical, physical, biological, and mineralogical compositionanalyses. The kurogaki trees had grown for more than 100 years in a mixture of relatively fresh andesiterocks and weathered rocks (Tomuro andesite) associated with dark brown soils. The pH of the soils isslightly acidic (6.4–6.8) at the foothills of Tomuro volcano in Kanazawa. The andesite rocks containlarge amounts of white plagioclase in 5-mm-sized, black small grains of hypersthene and amphibole,which originated from the eruptions of ash and lava 500,000 years ago.We identified and collected several trees with a black color on the branches, trunk, and roots andtheir associated andesitic soils, characterizing their structure and properties. At that time, the air radiationdose rate was 110–200 cpm, while BG was 70–80 cpm. The black “peacock pattern” portions showed asomewhat higher dosage, in the range of 150–200 cpm, based on the results from the Aloka β (γ) survey
Minerals 2017, 7, 1233 of 21
meter TSG-136. The black “peacock pattern” and beige portions from the taproot were compared withthe black root parts for the samples collected from Tawara in January 2017 (Figures 1 and 2).
Figure 1. The black “peacock pattern” of kurogaki wood is used for manufacturing various articlesof: furniture (a); and tea ceremony (b) in Japan. A view of 100-year-old kurogaki in December 2016,planted at Tawara, Ishikawa, Japan. Many roots (5–10 m in length, 30–50 cm in diameter) developed inall directions. A cut-down trunk is shown in (c) (176 cm in length and 40 cm in diameter). The blackdivision of the: vertical section (d); and cross section (e) in the trunk indicates a hard black part, shownby white arrows. The large root of 32 cm in diameter is divided into three smaller roots. A fragment ofthe Tomuro andesite rock is caught between the roots (arrow in (f)).
Views of cut-down kurogaki trees in the hilly Tawara ﬁeld are shown in the Figures. The thickand long roots hold the large stalk of kurogaki trees (Figure 1c), resulting in hollowing-out of the trunkrepresented by a black color, which reﬂects siliciﬁcation and ligniﬁcation (arrow in Figure 1d). There isalso a black injury on a 40-cm cross section (arrow in Figure 1e), and one andesitic rock is caughtbetween large roots in a 32-cm cross section (arrow in Figure 1f).
Minerals 2017, 7, 1234 of 21
2.2. H2O2 Reaction Checks
We tested the black and beige portions of the “peacock pattern” wood with a 3.5% H2O2 solution.If oxygen is generated upon adding H2O2 to a black part, this indicates the presence of organics;enzyme catalyst; metals such as Fe, Cu, Mn, and Cr; and microorganisms (arrow in Figure 2c).
Figure 2. The cross section of another shorter root, 50-cm in length and 20-cm in diameter. The whitearrow indicates the black spot at the cross section (a) the inside of the root is filled with black soft materials;(b) The black portions react with H2O2 solution, showing many white bubbles (an arrow in (c)), whichare about 1.00-mm in diameter. The black soft materials were analyzed by XRD, ICP-MS, and observedby SEM-EDS and TEM.
Many organisms can decompose hydrogen peroxide (H2O2) enzymatically. Enzymes are globularproteins, responsible for most of the chemical activities of living organisms. They act as catalysts,substances that speed up chemical reactions without being destroyed or altered during the process.H2O2 is toxic to most living organisms. Many organisms are capable of enzymatically destroying theH2O2 before it can do much damage. H2O2 can be converted to oxygen and water, as follows:
2H2O2 → 2H2O + O2(g)
2.3. X-ray Powder Diffraction
To identify minerals and analyze the structure of crystals and crystallinity of cellulose in thetrunk and the black roots of kurogaki (Figure 2b) associated with andesitic rocks Figures 5 and 6,we used X-ray powder diffraction analysis. An X-ray powder diffractometer (XRD) (Rinto 2200,Rigaku, Tokyo, Japan; Cu-Kα radiation, 40 kV, 30 mA, scan speed of 2◦/min) was used for identifyingminerals in kurogaki black roots at Tawara, Kanazawa, Japan. The powder samples (bulk, <2 µm, andsuspension) were used for X-ray powder diffraction analyses.
2.4. XRF Chemical Analyses
The kurogaki tree samples at Tawara were investigated using an X-ray ﬂuorescence analyzer(Rigaku Primus II, Tokyo, Japan), which operated at an accelerating voltage of 60 kV, 150 mA (max.),4 kW, under vacuum conditions, after heating (KDFS 80) at 600 ◦C for 30 min. The samples werepressed (Table 1).
Minerals 2017, 7, 1235 of 21
2.5. ICP-MS Analyses
The semi-quantitative analyses of black and beige portions of the kurogaki samples wereconducted using Elementar Analysensysteme GmbH Vario MAX type (ICP-MS), Thermo FisherScientiﬁc (Bremen, Germany) iCAP, and compared with the kurogaki sample collected from Tawaraand Makiyama, Kanazawa, Japan (Table 2).
2.6. Scanning Electron Microscopy (SEM) Equipped with Energy-Dispersive Spectroscopy (EDS)
A scanning electron microscope (S-3400N. Hitachi, Ibaragi, Japan) equipped with anenergy-dispersive X-ray analyzer (Horiba EMAX, Kyoto, Japan) was used to study the kurogakisamples collected from Tawara. We attempted to analyze the appearance, distribution, tissue structure,and inorganic and organic constituents of the wood, evaluating the morphology at the microscale.The conditions used were as follows: 15 kV accelerating voltage, 70–80 µA current, analytical time of1000 s, and an area of 10 mm × 10 mm on a carbon double tape with C coating (Figures 7–13).
2.7. Transmission Electron Microscopy (TEM)
A transmission electron microscopy (Hitachi H7650, Ibaragi, Japan) was used to study the blackparticles suspend in the water of kurogaki powder samples collected from Tawara. We attempted toobserve the appearance and morphology of grains, distribution, tissue structure with inorganics andmicroorganisms in the black parts, evaluating the morphology at the microscale. The conditions usedwere as follows: 80 kV accelerating voltage, on a micro grid without any coating (Figure 14).
3. Results
3.1. H2O2 Reaction
The black portion of the roots collected from Tawara reacted strongly with the 3.5% H2O2 solution,allows looking for organics (either dead organic materials or living one such as microbes) or manganese(arrow in Figure 2c). The beige region did not react with the H2O2 solution, showing no bubbles. It waspossible to divide catalysts into two portions, the black and beige parts, indicating biological catalystsof enzymes at black portions. Enzymes are proteins responsible for most of the chemical activities ofliving organisms at black portion of kurogaki.
3.2. Mineralogy of Kurogaki and Andesitic Rocks (Tomuro-ishi) around the Roots of Kurogaki
The mineralogy of the black parts at top trunk (Figure 3a), black parts at middle trunk (Figure 3b)and black parts at central root (Figure 3c) of the kurogaki samples from Tawara was determined usingXRD analyses. The black parts contained mainly crystalline cellulose ((C6H10O5)n; 5.54 Å, 5.41 Å)associated with kaolinite (Al2Si2O5·(OH)4·H2O (7.38–7.01 Å)), cristobalite (SiO2 (3.997, 3.630, 2.385,2.059 Å)) and apatite (Ca5(PO4)3(F, Cl, OH) (2.978 Å)). The peaks of cellulose indicated wider andgradual reﬂections, whereas those of cristobalite indicated sharp and strong reﬂections (Figure 3).The mineralogy of the black parts at trunk of kurogaki dead body (Figure 4a) and black parts atcentral root in Tawara (Figure 4b) samples was determined using XRD analyses (Figure 4b). The peaksof cellulose indicated wider and gradual reﬂections, whereas those of cristobalite, kaolinite (7.273 Å)and apatite (2.986 Å) indicated sharp and strong reﬂections (Figure 4a). Especially the kurogaki deadbody suggested mineralization of kaolinite, cristobalite and apatite in black parts of wood structure.
Minerals 2017, 7, 1236 of 21
Figure 3. The black parts at top trunk (a); black parts at middle trunk (b); and black parts at centralroot (c) were analyzed by XRD, indicating mainly crystalline cellulose (C6H10O5)n at 5.54 Å and 5.41 Årefractions, associated with kaolinite Al2Si2O5 (OH)4·H2O at 7.38–7.01 Å; cristobalite SiO2 at 3.997 Å,3.630 Å, 2.385 Å, and 2.059 Å; and apatite Ca5(PO4)3(F, Cl, OH) at 2.978 Å refractions.
Figure 4. The black parts at trunk of kurogaki dead body (a); and black parts at central root in Tawara(b) were analyzed by XRD, indicating mainly crystalline cellulose (C6H10O5)n at 5.460 Å associatedwith kaolinite Al2Si2O5 (OH)4·H2O at 7.273 Å; cristobalite SiO2 at 3.666 Å, 2.509 Å, and 2.360 Å; andapatite Ca5(PO4)3(F, Cl, OH) at 2.986 Å refractions ((a) fossilized black parts of kurogaki trunk; and(b) beginning of biomineralization of kurogaki root).
On the other hand, the relatively fresh andesite rocks (Figure 5a), the weathered rocks (Figure 5b),and soils with clays (Figure 5c) around the roots of kurogaki, as analyzed by XRD, were subjectedto mineralogical composition analyses during the weathering processes that affect kurogaki trees.The fresh andesite rocks (Figure 5a), called “Tomuro-ishi”, were produced from Mt. Tomuro volcano(Quaternary period; about 600,000–500,000 years ago). The andesite fresh rocks (Figure 5a), weatheredrocks (Figure 5b) and clayey soils (Figure 5c) are distributed widely throughout Kanazawa (Figure 5).
Minerals 2017, 7, 1237 of 21
Figure 5. Fresh (a); weathered (b); and clayey soils (c) Tomuro andesite rock samples are collected fromkurogaki root (Figure 1f) at Tawara, Ishikawa, Japan. The weathered andesite rock is sandwiched inthe roots, as shown in Figure 1f (an arrow).
The Tomuro fresh andesite rocks are composed of mainly feldspars (Orthoclase, Labradorite),mica (biotite), quartz, calcite, dolomite, cristobalite, and a trace of kaolinite, whereas the weatheredandesite is composed of higher contents of kaolinite and cristobalite than the fresh andesite. The soilsof weathered andesite are rich in clay minerals, such as 14 Å clays, kaolinite (7.2 Å), gibbsite, andcristobalite. The 4.2–4.4 Å reﬂection peak in the soils has increased intensity compared to that of freshand weathered andesite rocks, suggesting that the content of clay minerals is increased by weathering(Figures 5 and 6). The Tomuro ﬂesh andesite (1); weathered Tomuro andesite (2); and the soils (3)were composed of minerals, such as 14 Å clays, biotite (9.8 Å and 1.54 Å), feldspers (6.5 Å and 3.17 Å),kaolinite (4.2–4.4 Å, 2.5–2.6 Å, 1.82 Å, 1.54 Å, and 1.37 Å), cristobalite (4.0 Å and 2.12 Å), mica (3.4 Å),quartz (3.3 Å), calcite (3.0 Å), and dolomite (2.8 Å, 1.44 Å, and 1.40 Å), respectively (Figure 6).
Figure 6. XRD analyses of: fresh Tomuro andesite (1); weathered Tomuro andesite (2); and soils (3)near kurogaki roots (Figure 1f) at Tawara, Kanazawa, Ishikawa, Japan, showing a clear weatheringprocess to produce clay minerals of kaolinite and cristobalite near kurogaki roots.
Minerals 2017, 7, 1238 of 21
3.3. XRF Chemical Analyses
The chemical composition o: fresh Tomuro volcanic fresh andesite (1); weathered andesite (2); andsoils (3) collected from near kurogaki roots at Tawara, Ishikawa (Figure 5) showed the major and traceelements by XRF analyses (Table 1). The elements of SiO2, Al2O3, Fe2O3, CO2, CaO, Na2O, K2O, andMgO are major elements of andesite, associated with trace elements of TiO2, P2O5, MnO, WO3, SrO,and SO3. The trace elements are attributed to organics, such as microorganisms, showing SEM-EDSelemental content maps in Figures 7–9. Some of elements decreased by weathering, such as SiO2, CaO,Na2O, MgO, WO3, SrO and CuO.
Table 1. XRF analyses of Tomuro volcanic andesite (quaternary period) near kurogaki roots at Tawara,Kanazawa, Japan, showing weathering processes from fresh rocks to weathered rocks and fertile soilsnear kurogaki roots at microbial communities. (mass %)
ElementsFreshWeatheredSoilsBy Weathering
SiO262.000060.100055.6000>decreasedAl2O317.300021.100019.3000<Fe2O35.24005.37006.0800<CO24.22004.990012.3000<CaO3.76002.60001.0600>decreasedNa2O3.01001.64001.2000>decreasedK2O2.14001.61002.0800=MgO1.33001.16001.0700>decreasedTiO20.46400.50100.6580<P2O50.29900.54500.2230=MnO0.13800.14400.1120=WO30.04030.02060.0156>decreasedSrO0.03820.02220.0166>decreasedSO30.03670.11500.0957=V2O50.0155ZrO20.01260.01230.0186<ZnO0.00880.0087=Rb2O0.00770.00920.0085=Co2O30.00590.00700.0076<Cl0.0073NiO0.00300.0032CuO0.00190.00190.0015>decreasedY2O30.00150.00160.0017<Ga2O30.00180.0031<PbO0.00150.00100.0030<Nb2O50.00170.0018<Br0.00140.0038<BaO0.0428ReO20.0325SnO20.0194I0.0147Cl0.0073Ga2O30.0031Nb2O50.0018As2O30.00130.0009>decreased
3.4. ICP-MS Analyses of Kurogaki
ICP-MS analyses indicated the presence of major and trace elements in the chemical compositionof the black and beige portions of the kurogaki samples collected from Tawara, in comparison withthe samples collected from Makiyama (Table 2). Nearly all elements were more abundant in the blackportions of the wood than the elements in the beige portions.In particular, Ca (29,200 mg/kg), Mg (6370 mg/kg), K (2160 mg/kg), P (701 mg/kg),Mn (575 mg/kg), Ba (462 mg/kg), S (576 mg/kg), Cl (392 mg/kg), Fe (326 mg/kg), Na (182 mg/kg),and Al (151 mg/kg) contents were high in the black region. Trace elements such as B, Si, Ni, Sr,Ti, Zn, Cu, Pb, and Rb could also be seen in the black portions of kurogaki samples collected fromTawara. The chemical composition in the black region of the samples collected from Makiyamaindicated a similar tendency for main elements, in that they were more abundant in the black portions
Minerals 2017, 7, 1239 of 21
than in the beige ones. Note that the concentrations of B and Ba are important characteristics incolored persimmon.
Table 2. ICP-MS elemental analyses of kurogaki at Tawara and Makiyama, involving comparison ofbeige and black portions, indicating that Ca, Mg, S, K, P, Mn, Ba, Cl, Fe, Na, and Al are present at highdensity in the black region.
PlaceTawaraMakiyamaMakiyama
RegionBlackBlackBeigeElementsmg/kgmg/kgmg/kg39K21604900341044Ca2920024100134035Cl3921110109024Mg6370281066934S576188057227Al151223052456Fe32685237531P70161132623Na18217612055Mn5754294511B4711521137Ba4621851229Si7860Ni7088Sr6948Ti4966Zn4063Cu29208Pb2285Rb5
3.5. Scanning Electron Microscopy (SEM) Equipped with Energy-Dispersive Spectroscopy (EDS)
SEM observation of the black pith in the central root at Tawara showed various typedmicroorganisms with different sizes from several tens of micrometers to 500 µm in size (Figures 7 and 8).The microorganisms showed different shapes, such as coccus-type and filamentous bacteria, indicatingfission process. The fission of coccus-type bacteria can be seen in Figure 7a–d. The bead-like microorganismsare commonly present in the black portion (Figure 7e,f). Development processes of broomstick-likemicroorganisms from the root to the very top of the stick can be seen in Figure 8c,d; some of themapproach 300 µm or more in length.
Figure 7. Cont.
Minerals 2017, 7, 12310 of 21
Figure 7. SEM micromorphology of black pith in the central root at Tawara (Figure 2b) showing:coccus and ﬁlamentous bacteria (a–d); and wooden clapper microorganisms (e,f). The developingﬁlamentous bacteria drawn lengthways can be seen in (b,c). The cell division of coccus-type bacteriacan be seen in (a,b,d).
Figure 8. Cont.
Minerals 2017, 7, 12311 of 21
Figure 8. SEM micromorphology of black pith in the central root at Tawara (Figure 2b) showingbroom-type bacteria (a–d). The rudiment of a broom is less than 20 µm in length (b), the broomstretches easily from several tens of micrometers to 500 µm in length (c,d). Material like the broomseems to grow from holes on the sheeting (a,b), showing the root portion (c) and the top of the broom (d).
3.6. SEM-EDS Analyses of Kurogaki
SEM-EDS semi-quantitative analyses of kurogaki for four different points of the black portionsof the taproot collected from Tawara revealed the accumulation of particular elements at each point(Table 3). The highest concentrations were of P (41–50 atomic content %) and Ca (17–26 atomic content %),suggesting the formation of phosphorus carbonate, such as apatite, which is agreed with XRD data(Figures 3 and 4). Small amounts of S (6.4–7.3 atomic content %), Mn (3.4–4.6 atomic content %),Mg (3.5–5.1 atomic content %), and Si, K, Fe, and As were also detected.On the other hand,Sr (5.0–5.9 atomic content %) and Pb (9.0–10.2 atomic content %) were notable for their high content inthe black portions (Table 3).Elemental content maps of points 2–4 in Table 3 are shown in Figures 9–11, respectively.The presence of various microorganisms, such as coccus-type bacteria, ﬁlamentous bacteria,broom-type bacteria, and a pair of beaded bacteria, was associated with high concentrations ofMg, Si, P, S, Cl, K, Ca, Mn, Fe, Sr, and Pb in the black portions of the taproot (Figures 9–11). However,the Cd, Sr, Mn, Fe, and Pb elements dotted among the map.The ﬁlamentous and coccus-type bacteria exhibited high P and Ca concentrations, suggesting theformation of carbonates in the black parts of the taproot. In addition, the concentrations of Sr and Pbwere extremely high at points 1–4, suggesting that the kurogaki samples obtained from Tawara arehyperaccumulators of these elements [1,2].
Table 3. SEM-EDS semi-quantitative analyses of black portions at the central root of kurogaki inTawara, indicating high concentrations of P, Ca, S, Pb, and Sr (Figure 2b). The elemental content mapsare shown in Figure 9 (point 2), Figure 10 (point 3), and Figure 11 (point 4).
ElementsPoint 1Point 2Point 3Point 4
Mg Kα13.683.515.122.77Si Kα10.830.101.201.10P Kα145.5650.4640.9046.47S Kα16.846.407.287.33Cl Kα10.000.000.000.25K Kα11.001.321.431.33Ca Kα123.0717.1025.9719.81Mn Kα13.734.553.444.25Fe Kα10.260.470.630.22As Lα10.040.060.060.27Sr Lα15.445.875.045.88Pb Mα9.4610.168.9410.35Total%99.91100.00100.01100.03ContentFigure 9Figure 10Figure 11
Atomic content (%): ±3σ.
Minerals 2017, 7, 12312 of 21
Figure 9. SEM-EDS image and elemental content map of black portions at the central root at Tawara(Figure 2b), showing broom-type bacteria on the wood formative tissue associated with the elementsMg, Si, P, S, Cl, Cd, and Pb. The analytical data of this elemental content map are shown in Table 3(point 2).
Figure 10. SEM-EDS image and elemental content map of black portions at the central root (Figure 2b),showing ﬁlamentous bacteria associated with Si-Sr mineral grains (arrow). The analytical data of thiselemental content map are shown in Table 3 (point 3). Si and Sr are distributed over the same place(arrows), showing high concentrations of Mg, P, S, and Ca.
Minerals 2017, 7, 12313 of 21
Figure 11. SEM-EDS image and elemental content map of black portions at the central root (Figure 2b),showing coccus bacteria with Si-Sr mineral grains (arrows) associated with carbonate minerals.The analytical data of this elemental content map are shown in Table 3 (point 4). Si and Sr aredistributed over the same place (arrows). The concentrations of Mn, Fe, and As are very low, whereasboth Sr and Pb are concentrated.
3.7. SEM-EDS Elemental Content Maps of Kurogaki
The elemental content maps of black portions at central root of kurogaki at Tawara, showeddistribution of bio-minerals associated with microorganisms, such as silicate minerals and carbonateminerals (Figures 12 and 13). The combination of elements such as Si, Sr and Ca accords with the otheranalytical results of XRF and IC-Mass.
Figure 12. SEM-EDS image and elemental content map of black portions at the central root at Tawara(Figure 2b), showing cocuss typed bacteria associated with Si–Sr–Ca mineral grains with 3 µm–10 µmin diameter (arrows and circles). The small Ca grains suggested amorphous Ca carbonate.
Minerals 2017, 7, 12314 of 21
Figure 13. SEM-EDS image and elemental content map of black portions at the central root at Tawara(Figure 2b), showing ﬁlamentous bacteria associated with Ca-rich grains (arrows). The Ca-rich grainssuggested low crystalline Ca carbonate minerals before apatite mineral.
3.8. TEM Observation of Black Parts of Kurogaki
TEM observation of kurogaki black parts of roots in Tawara shows various kind of particles, suchas thick chunks of grains (cellulose), precursor thin ﬁlms, angular and square (cristobalite), and varioussized and shaped microorganisms (coccus type, Bacillus type, and ﬁlamentous bacteria, few microns insize) (Figure 14a,b). Some of precursor thin ﬁlms may be clay minerals associated with thick celluloseand bacteria (Figure 14c,d). We did not obtain electron diffraction patterns of each particle, whichmight be future study.
Figure 14. TEM micromorphology of black parts of kurogaki root at Tawara, showing cellulose,precursor thin ﬁlms, bacteria, and cristobalite grains. (a) Bacteria and precursor ﬁlms, (b) bacteriaand cellulose, (c) bacteria, cellulose and precursor ﬁlms, (d) bacteria, thin ﬁlms of cellulose, andcristobalite grains.
Minerals 2017, 7, 12315 of 21
4. Discussion
The bark tissues can be divided into two major regions: the inner bark and the outer bark.Physiologically, the inner bark transports the assimilates and serves as a storage organ for food reserves,while the outer bark is physiologically inactive and forms a protective layer against mechanical andchemical injury. Accordingly, basic chemical studies on bark are important not only to understand thephysiology of trees but also to ensure better utilization of the bark [1]. From this viewpoint, the chemicaland physical properties of bark lignin and bark phenolic compounds have been investigated [2–5].On the other hand, the heartwood of the Japanese persimmon tree (Diospyros kaki) becomes blackon rare occasions and has been highly sought after as a substitute for ebony. Previous research hasattempted to clarify how the physical, mechanical, chemical, and biodegradation properties differbetween lighter-colored sapwood and blackened heartwood in D. kaki. The speciﬁc gravity, equilibriummoisture content, modulus of rupture, and modulus of elasticity in the blackened heartwood have beenshown to be higher and the loss tangent lower than those in sapwood [5]. However, these previousstudies did not address biomineralization of the wood structure.In this paper, we report the characterization of not only trunk tissue but also root tissue of kurogakiassociated with the striking black “peacock pattern” at Kanazawa, Ishikawa, Japan, based on radioactivity,ultraviolet analysis, and H2O2 reactions in the field. We also report for the first time the results ofbiomineralization using ICP-MS, XRD, XRF, SEM-EDS and TEM. These results can provide insights intothe simple identification of mineral species and characteristic structures (Figure 15). We analyzed themineralogy, chemistry, and micro-morphology of tissues by using a combination of micro-techniques.The black portions were found to consist mainly of cellulose, high-crystalline cristobalite and apatite,associated with kaolinite and many microorganisms (Figure 16). Particular elements such as abundantCa, associated with Mg, Si, P, S, Cl, and K, were accumulated at the same sites as microorganisms,as shown in the elemental content maps (Figures 9–13).The compositions of black and beigeportions of the black persimmon tree were determined based on chemical data obtained from ICP-MSanalyses (Table 2, Figure 15).The objective of this study was to illustrate the potential role of various microorganisms in themineral composition of black persimmon. Microorganisms have the ability to incorporate silicon intovarious organic compounds, forming C–O–Si linkages and more complicated hard structures.
4.1. Formation of Crystals and Amorfous Materials in the Trunk and Roots
The mineral materials in the trunk proved that they consist of cristobalite, apatite and kaolinite byXRD, whereas amorphous calcium carbonate and calcium oxalate existing in living cells at the blackportions of central root, namely the bacteria by SEM-EDS. This is the factor attributed to the growth ofhigh crystals from low crystals or amorphous calcium carbonate in the annual rings. The cristobaliteand apatite, in particular, is formed within the living cells in association with bacteria; this resultsin increased hardness of the trunk. Microorganisms are thus an integral part of biomineralizationwithin wood tissues of kurogaki. Microbial communities of phytostabilization are extremely complex,containing thousands of species associated with both light and heavy elements, and the exact functionof most species, in a given environment, is simply illustrated the points with diagrams andesiteweathering soils—kurogaki root tissue and trunk tissue with the striking black “peacock pattern” inkurogaki (Figures 15 and 16).
Minerals 2017, 7, 12316 of 21
Figure 15. Concentrated nutrient elements for the black region in kurogaki roots and andesitic soils,using ICP-MS (red), XRF (green), and SEM-EDS (black).
Figure 16. Schematic formation processes of the peacock pattern at trunk cross section and blackportion in roots of black persimmon tree at Tawara, Kanazawa, Japan, showing biomineralizationand phytostabilization at kurogaki roots, associated with Tomuro andesite weathering processes atmicrobial communities with oxalic acid.
In contrast to kurogaki, doronoki (Populus maximowiczii) grows rapidly and the wood is softand light. It has recently been planted in South Korea and Japan for use as pulpwood, lumber,boxes, matches, and other miscellaneous articles. However, when the wood is processed, the edgeof the sawing machinery and tools is easily worn away and spoiled due to the crystals of inorganicmatter contained in the wood [6]. The crystals in the heartwood are primarily white. In the caseof the discolored wet wood of P. maximowiczii, crystals and certain anaerobic bacteria are observed.The mineral content of the sap is the highest during fall, and the crystals prove to be calcite of calciumcarbonate and calcium oxalate existing within living cells of bacteria. This can be the reason why thecrystals grow in a ring-like pattern in the late wet wood formed during the year [7–9].
Minerals 2017, 7, 12317 of 21
Strontium carbonate is more insoluble than calcium carbonate and precipitates under appropriateconditions. Ferris et al. (1995) observed the precipitation of strontium-rich calcite on a serpentineoutcrop in a groundwater discharge zone near Rock Creek, British Columbia, Canada [10].These mineral precipitates nucleate around epilithic cyanobacteria, including Colothrix, Synechococcus,and Gloeocapsa. The strontium content of calcite is as high as 1 wt % and strontium carbonate formsa homogeneous solid solution in calcite. Intracellular Sr-Mg-Ca carbonate also precipitates in the cellsof some cyanobacteria from an alkaline lake in Mexico [10].
4.2. Formation of Minerals in the Soils near Kurogaki Roots
Weathered Tomuro andesite changes to fertile soils under conditions of pH 6.4–6.8, containing clayminerals of kaolinite and cristobalite. Weathering of Tomura andesite rocks dissolves the constituentparts of the rocks (SiO2, CaO, Na2O, MgO, TiO2, WO3, and SrO) (Table 1). Microorganisms in thefertile soils mediate between soils and roots of kurogaki trees, associated with calcium oxalate (CaC2O4or (COO)2Ca), which does not mix with soils and water showing SEM-EDS crystals. Plants soak up thedissolved elements from rocks. At the rudiments of micron-scale biomineralization of kaoliniteand cristobalite occurred in kurogaki root during Tomuro andesite weathering. The black andbeige coloration, referred to as a “peacock pattern”, then emerges at the trunk of kurogaki afterbiomineralization around the roots (Figures 14–16).The weathering process has generally been considered from only a chemical/physical perspective;however, recent observations of bacteria in weathered rocks have questioned the importance ofmicrobial activity in this process. To examine this, an outdoor natural experiment was performed,where Tomuro andesite rocks were immersed in running groundwater at outside temperature forone year [11,12]. After three months of incubation in the running groundwater, biomineralizationof carbonate minerals (calcite and aragonite) by cyanobacteria was found. After one year, bio-clays(smectite) and zeolite (heulandite and clinoptilolite) were found. Various microorganisms, such ascyanobacteria, diatoms, and bacteria, accelerated the weathering reactions of Tomuro andesite.Microorganisms play an important role in the change of soils from andesite within such a shortperiod, showing the accumulation of elements such as Si and Ca, which could have been derived bydissolution of the rocks associated with rainwater.In this study, the XRF, ICP-MS, XRD, SEM-EDS, and TEM data collectively demonstratethe microbial formation of the bio-clays kaolinite, apatite, and crystobalite near kurogaki roots.The microorganism activity near the roots may hence have a great inﬂuence on the clay mineraldevelopment commonly observed in naturally weathered rocks. Characteristics of soils, fertilizer,agriculture, and forestry are biological production using ecosystem services in natural and/or artiﬁcialﬁelds. In these ﬁelds, the roles of clays and other inorganic materials involve providing essentialnutrients to plants and animals, and to conserve the environmental conditions suitable for their growthand sustenance [9].Some plants that accumulate heavy metals have been reported. For example, Thlaspi calaminareaccumulates Zn, Brassica juncea accumulates Pb, and Populus accumulates TCE at the rhizosphere,which is the most active site of living microorganisms in the soil. Biomethylation can be performed withAs, Hg, Cd, and Pb of remedial options for metal-contamination sites near plants [13,14]. In this study,some of the essential elements were transported to kurogaki roots via soils from weathered andesiterocks (Tomuro-ishi) by bacterial biomineralization, as shown by the XRF, ICP-MS, XRD, and SEM-EDSanalyses. Especially in the SEM-EDS results, elemental content maps of the black portions of kurogakiindicated higher populations of microorganisms than in the beige areas, suggesting biomineralizationprocesses (Figures 7 and 16). Biomineralization started in the areas of kurogaki taproot, and developedminerals, such as cristobalite, apatite, strontinite, carbonate, and kaolinite clays, in the black parts ofthe root, which proceeded to substitute elements as follows:
Ca —– → Sr, Ba, P—- → Si, CO3 — → PO4,
Minerals 2017, 7, 12318 of 21
The black portions were more resistant to fungal and termite attacks than the beige portions.The mineralization process is thus speculated to be a defense mechanism. These results will be of usenot only in scientiﬁc research but also for the advancement of important materials for manufacturingfurniture and miscellaneous articles in Japan, potentially contributing to the vitality of local economies.
4.3. Physical and Chemical Characteristics of the Black Portion
Wood color parameters showed differing relationships with soil chemical properties, rangingfrom no relationship to a weak or moderate relationship. For instance, soil pH decreased moderatelywith decreasing L* values (increasing darkness), and there was very little evidence that wood color wasinﬂuenced by soil exchangeable cations (Ca2+, Mg2+, Na+, and K+). However, results from previousstudies [9] disagree with these ﬁndings. They showed that yellowness (b*) was more affected by soilor ecological zones. However, redness (a*) and yellowness (b*) are not good indicators of durabilityin teak wood because they are related to the species of fungi [13]. Microcodium root calciﬁcationproducts of terrestrial plants on carbonate-rich substrates have been revisited elsewhere [12,15].In this study, ICP-MS analytical data indicated higher concentrations of Ba (16 times) andB (6.7 times) in the black parts than in the beige parts (Table 2). The ﬁndings of trace elementanalysis using ICP-MS also showed that the boron content was markedly higher in the black portions.This suggests that boron has antifungal properties associated with the blackening of Japanesepersimmon [3]. Although many techniques to standardize color in wood have been developed,such as drying schedules or heat treatment, as well as the application of chemical substances, studieson heartwood color control in teak are limited. For this reason, control of color of teakwood should beproperly addressed in future research.Matsushiro Hot Spring in Nagano, Japan, is known for its very high concentration of boron(854 mg B/kg). The microbial mat there is more than 2 m in depth and contains more than 50 wt % Ca,indicating the presence of calcite. Cyanobacteria and diatoms inhabit these black microbial mats.Cyanobacteria use an enzyme named carbonate anhydrase (CA), which breaks down the HCO3−
dissolved in the water into CO2 and OH− [16].
4.4. Relationships among Microorganisms, Calcite, and Trace Elements in the Black Portion of Kurogaki
The traces of not only B and Ba but also Sr can be ascribed to an association of abundantmicroorganisms with the calcification and color of black persimmon in the severe environment, indicatinga capacity for absorbing both radionuclides and stable isotope elements.Microbial precipitationof strontium calcite in hot springs and a groundwater discharge zone has been reported [17,18].Filamentous fungi, yeasts, algae, bacteria, and diatoms have been evaluated for their ability toremove radioactive elements via bio-sorption. They can accumulate B, Ba, Sr, and radionuclidesthrough precipitation and complexation on and within environments containing hydroxyl and carboxylgroups. Calciﬁcation and diagenesis of bacterial colonies, the role of fungi in the biomineralization ofcalcite, and an early-branching microbialite cyanobacterium forming intracellular carbonates werereported by many researchers [18–21]. These various microorganisms live in soil and water, producingdistinct minerals such as barite (BaSO4), celestite (SrSO4), and gypsum (CaSO4·2H2O). However,biomineralization in living wood, such as kurogaki, has never been reported.
4.5. Bacterial Precipitation of Calcium Carbonate
The Ca in calcium oxalate (CaC2O4 or (COO)2Ca) and Ca5(PO4)3(F, Cl, OH) in the taproot ofkurogaki can easily be substituted with Sr, Ba, Ce and Pb, whereas P can be substituted with As, V,and Si. In addition, CO3 can be partially substituted with PO4 in the roots (Figures 9–13). Moreover,the Sr in SrCO3 can be substituted with Ca, Ba and Pb through precipitation and complexation onand within the environments containing hydroxyl and carboxyl groups with bacterial colonies in thehyper accumulator [22–28]. Typical CaCO3 minerals, such as calcite and aragonite were not found intrunk and root of kurogaki, using XRD method, whereas high concentration of Ca and P were found
Minerals 2017, 7, 12319 of 21
by SEM-EDS and XRD methods which is formation of apatite in black parts at trunk of kurogaki.Bacterial precipitation of amorphous Calcium Carbonate is quite common at the microbial mats ofmodern and ancient microorganisms in stratiﬁed systems [29–31].
4.6. Biomineralization of Crystoballite Identiﬁcation
Formation of crystoballite by biomineralization in kurogaki is a new discovery, conﬁrmed bychemical, physical and biological data, using ICP-MS, XRF, XRD, SEM-EDS and TEM in this study.All the data put together, forming an idea of biomineralization between black parts sections, originatedminerals and microorganisms of black persimmon tree (Table 4). The exact nature of the naturalcrystobalite in kurogaki and their relationship between wood tissue and andesitic soils, however,requires further study.On the other hand, XRD combined with TEM/HRTEM techniques conﬁrmed crystalline characterto gain insight into the structural nature of the crystoballite, tridymite, and opals materials. To obtainmore direct evidence for the presence of two layer types in opal-CT, HRTEM images were reported forseveral samples [26–31].
Table 4. Biomineralization of Black Persimmon tree.
Black Parts SectionsOriginated MineralsMicroorganisms
Dead body of trunkKaolinite, Cristobalite, Apatite >>> CelluloseQuantities of bacteriaTop trunkKaolinite, Cristobalite, Apatite >> CelluloseQuantities of bacteriaMiddle trunkSmall amounts of kaolinite, Cristobalite > CelluloseQuantities of bacteriaCentral rootCellulose, CristoballiteQuantities of bacteriaat the top of the rootCellulose, CristoballiteQuantities of bacteria
Content Figures 2–4, Figures 7–14 and Tables 1–3.Kaolinite; Al2Si2O5(OH)4·H2O. Cellulose; (C6H10O5)n.Cristobalite; SiO2. Apatite; Ca5(PO4)3(F, Cl, OH).
5. Concluding Remarks
Although patterned kurogaki occurs in only one of every 1000–10,000 trees, we found severalspecimens at Tawara, Kanazawa, Japan, in 2016. We concluded that kurogaki microbiota are frommicroorganisms in the soil environment associated with siliciﬁcation and carbonate precipitation.Patterned kurogaki thus consists of siliciﬁed wood.This study is probably the ﬁrst to identify that the minerals in the black portions of kurogakitaproot and trunk contain not only cellulose but also kaolinite, cristobalite and apatite associated withmany microorganisms.Particular elements such as abundant P and Ca were found to be associated with Mg, Si, S, K, Pb,and Sr with small amounts of Cl, Mn, and Fe elements concentrated in the black portions, accompaniedby various microorganisms, as shown in SEM-EDS elemental content maps, associated with TEM andXRD analyses.Various microorganisms responded with a metabolic reaction of the roots of kurogaki, formingnot only cellulose but also CaC2O4 or (COO)2Ca in andesitic soils (pH 6.4–6.8).The compositions of black and beige portions of the black persimmon tree were determined basedon ICP-MS analyses. Almost all elemental contents in the black portions were higher than those in thebeige parts.The objective of this study was to illustrate the ability of various microorganisms associated withbiominerals that are capable of absorbing these elements (Na, Mg, Al, Si, P, S, Cl, K, Ca, Mn, Fe, Sr, Ba,and Pb) from soils and weathered andesite, as revealed by XRD and XRF analyses of Tomuro andesiteat Tawara, Kanazawa, Japan. SEM and TEM images showed various microorganisms propagating andundertaking cell division and a diffuse distribution of coccus-type bacteria, ﬁlamentous bacteria, andbroom-type bacteria in the black portions of kurogaki taproot. H2O2 solution showed reactivity in theblack portions, while the beige parts showed no reaction.
Minerals 2017, 7, 12320 of 21
In conclusion, the crystals of kaolinite, cristobalite and apatite associated with cellulose grewchemically and biologically in the sap under the conditions found in the taproot and trunk. We describethe existence of various microorganisms and other crystals including calcium, sulfur, and phosphorusoxalate existing in the living cells. This could be the reason why the crystals that grew in kurogakitaproot formed late during the year. The minerals are thought to have formed antigenically as a resultof the mineralization of organic matter by microbes.
Acknowledgments: We are grateful for the support from Wakura Co. (Yusuke Katayama). The authors also wishto thank Daisuke Seo, Azuma Taoka, Sachiko Yamamoto, Kiyoko Kuze, Hirono Tazaki, Hisashi Takahashi, andTomi Matsuba for their kind support in this research. The authors would also like to thank Enago (www.enago.jp;designed the Figures and Tables) for the English language review. We express our gratitude to Referees 1 and 2 fortheir helpful comments and advice on our work.
Author Contributions: K.T., Y.H., and S.H. conceived and designed the ﬁeldwork and sampling; K.N., M.O. andK.T. performed the XRD, XRF, and ICP-MS experiments; K.T. and T.T. operated the SEM-EDS and TEM; K.T., A.F.,and F.T. interpreted the data and wrote the manuscript; K.T., A.F., and F.T. designed the Figures and tables.
Conﬂicts of Interest: The authors declare no conﬂicts of interest.
References
1.Tazaki, K.; Takehara, T.; Hashida, Y.; Hashida, S.; Nakamura, K.; Yokoyama, A.; Aoki, C.; Tazaki, F.Physical and chemical characteristics and biomineralization of “Scarcity Black Persimmon Tree”. Earth Sci.2017, 71, 97–113.
2.Baker, A.J.M.; Mc Grat, S.P.; Reeves, R.D.; Smith, J.A.C. Phytoremediation of Contaminated Soils and Water;Terry, N., Banuelos, G.S., Eds.; CRC Press Inc.: Boca Raton, Florida, USA, 2000; pp. 85–107.
3.Sogo, M. Studies on the bark lignin and bark phenolic compounds. Mem. Fac. Agric. Kagawa Univ. 1971, 25,1–73.
4.Nakano, J. Chemical structure and color of lignin. Soc. Mater. Sci. Jpn. 1967, 16, 34–43. [CrossRef]
5.Noda, E.; Aoki, T.; Minato, K. Physical and chemical characteristics of the blackened portion of Japanesepersimmon (Diospyros kaki). J. Wood Sci. 2002, 48, 245–249. [CrossRef]
6.Lee, K. Crystals and their growth in the wood of Populus maximowiczii. Hokkaido Univ. Collect. Sch. Acad. Pap.1988, 43, 717–788.
7.Lukmandaru, G.; Ashitani, T.; Takahashi, K. Color and chemical characterization of partially black-streakedheart-wood in teak (Tectona grandis). J. For. Res. 2009, 20, 377–380. [CrossRef]
8.Moya, R.; Marin, J.D.; Murillo, O.; Leandro, L. Wood physical properties, color, decay resistance and stiffnessin Tectona grandis clones with evidence of genetic control. Silvae Genet. 2013, 62, 142–152. [CrossRef]
9.Nanzyo, M. Role of clays and other inorganic materials in soils, fertilizers, agriculture and forestry. Clay Sci.Soc. Jpn. 2017, 55, 88–96.
10.Ferris, F.G.; Fratton, C.M.; Gerits, J.P.; Schultze-Lam, S.; Lollar, B.S. Microbial precipitation of a strontiumcalcite phase at a groundwater discharge zone near Rock Creek, British Columbia, Canada. Geomicrobiology J.1995, 13, 57–67. [CrossRef]
11.Coureadeau, E.; Benzerara, K.; Gerard, E.; Moreira, D.; Bernard, S.; Brown, G.E., Jr.; Lopez-Garcia, P.An early-branching microbialite cyanobacterium forms intracellular carbonates. Science 2012, 336, 459–462.[CrossRef] [PubMed]
12.Tazaki, K. Formation of microbial mats and bio-clays at footbath- and rockyhill-outdoor systems. Clay Sci.2008, 47, 240–254.
13.Moya, R.; Calvo-Alvarado, J. Variation of wood color parameters of Tectona grandis and its relationship withphysical environmental factors. Ann. Sci. 2012, 69, 947–959. [CrossRef]
14.Kokutse, A.D.; Stokes, A.; Bailleres, H.; Kokou, K.; Baudasse, C. Decay resistance of Togolese teak(Tectona grandis) heartwood and relationship with colour. Trees Struct. Funct. 2006, 20, 219–223. [CrossRef]
15.Kosir, A. Microcodium revisited: Root calciﬁcation products of terrestrial plants on carbonate-rich substrates.J. Sediment. Res. 2004, 74, 845–857. [CrossRef]
16.Sato, K.; Tazaki, K.; Okuno, M.; Kubo, H. The microbes purify high concentration of metabolic acid water onthe microbial mats of Matsushiro Hot Springs, Nagano Prefecture, Japan. Earth Sci. (Chikyu Kagaku) 2010, 64,63–75.
Minerals 2017, 7, 12321 of 21
17.Tazaki, K.; Shimojima, Y.; Takehara, T.; Nakano, M. Formation of microbial mats and salt in radioactivepaddy soils in Fukushima, Japan. Minerals 2015, 5, 849–862. [CrossRef]
18.Tazaki, K.; Tazaki, F.; Okuno, M.; Takehara, T.; Ishigaki, Y.; Nakagawa, H. Formation of pisoliths at hotsprings in Saturnia, Toscana, Italy. J. Geol. Soc. Jpn. 2016, 122, 45–60. [CrossRef]
19.Robin, N.; Bermard, S.; Miot, I.; Blanc-Valleron, M.M.; Charbonnier, S.; Petit, G. Calciﬁcation and diagenesisof bacterial colonies. Minerals 2015, 5, 488–506. [CrossRef]
20.Bindschedler, S.; Cailleau, G.; Verrecchia, E. Role of Fungi in the biomineralization of calcite. Minerals 2016,6, 41. [CrossRef]
21.Saskia, B.; Guillaume, C.; Eric, V. Role of Fungi in the Biomineralization of Calcite. Minerals 2016, 61, 41.[CrossRef]
22.Morikawa, H.; Takahashi, M.; Kawamura, Y. New progress in environmental biotechnology usingphytoremediation. J. Environ. Biotechnol. 2001, 1, 1–14.
23.Jessica, M.E.; Stephen, B.R. TEM and X-ray diffraction evidence for cristobalite and tridymite stackingsequences in opal. Clay Clay Miner. 1996, 44, 492–500.
24.Malo, S.; Perez, O.; Hervieu, M. Spherulite-shaped cristobalite by fused silica devitriﬁcation. J. Cryst. Growth2011, 324, 268–273. [CrossRef]
25.Kopani, M.; Kopaniova, A.; Trunka, M.; Caplovicova, M.; Rychly, B.; Jakubovsky, J. Cristobalite and HematiteParticles in Human Brain. Biol. Trace Elem. Res. 2016, 174, 52–57. [CrossRef] [PubMed]
26.Withers, R.L.; Tompson, J.G.; Welberry, T.R. The structure and Microstructure of α-Cristobalite and ItsRelationship to β-Cristobalite. Phys. Chem. Miner. 1989, 16, 517–523. [CrossRef]
27.Masciocchi, N.; Cairati, P.; Sironi, A. Crystal structure determination of molecular compounds fromconventional powder diffraction data: Trimeric silver (I) 3, 5-dimethylpyrazolate. Powder Diffr. 1998,13, 35–40. [CrossRef]
28.Hassan, N.; Verdinelli, V.; Ruso, J.R.; Messina, P.V. Mimicking Natural Fibrous Structures of Opals by Meansof a Microemulsion-Mediated Hydrothermal Method. Langmuir 2011, 27, 8905–8912. [CrossRef] [PubMed]
29.Simkiss, K.; Wilbur, K.M. Biomineralization: Cell Biology and Mineral Deposition; Academic Press, Inc.:Waltham, MA, USA, 1989; p. 337.
30.Krumbein, W.E. Microbial Geochemistry; Blackwell Scientiﬁc Publications: Oxford, UK, 1983; p. 330.
31.Seckbach, J.; Oren, A. Microbial Mats: Modern and Ancient Microorganisms in Stratiﬁed Systems; Springer:New York, NY, USA, 2010; p. 606.
© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).