資料來源: NaturWissenschaften (Springer-Verlag 2001) PDF檔(有圖)請洽維中 Javier Garcia-Guinea · Victor Cardenes Angel T. Martinez · Maria Jesus Martinez Received: 31 May 2001 Accepted in revised form: 23 June 2001 Published online: 17 July 2001 Fungal bioturbation paths in a compact disk Abstract We report here on bioturbation traces, with micro-dendrite textures, composed of a mixture of altered aluminum and polycarbonate, which have been developed in a common compact disk (CD), destroying information pits. Fungal hyphae proliferated in these deteriorated zones, and Geotrichum-type fungus was isolated from surface-sterilized CD fragments. The severe biodeterioration described is attributed to the slow growth of this arthroconidial fungus on the CD material in the tropical indoor environment of Belize, Central America (~30°C, ~90% humidity). 亞佛‧賈西-朱尼亞, 維克多‧卡登尼斯 安琪拉‧T‧馬丁尼茲, 瑪利亞‧耶穌‧馬丁尼茲 (這名字也太... Orz) 接稿日: 2001年5月31日 最終修正版收稿日: 2001年6月23日 上線發行日: 2001年7月17日 CD片上的真菌生物擾動軌跡 摘要 在此我們要報告的,是一種在普通CD上發展,由變質的鋁和聚碳酸酯組成,且呈現微 樹突狀組織的生物擾動作用痕跡,及其對CD資訊槽所造成的破壞. 真菌菌絲在變質的區域 增生 -- 消毒過的CD碎片表面,分離出了某種地絲菌型的真菌. 這嚴峻的生物腐蝕,正是 貝里斯的熱帶環境中(室內,~30°C,~90%相對溼度),該節-分生胞子型的真菌於CD上緩慢 生長所致. Introduction The fungus Geotrichum was described more than 175 years ago as a white hyphomycete with hyphae disarticulating into cylindrical spores. It is commonly found in close proximity to human life. Geotrichum species are present in foodstuffs such as cereals, cheese, yeast and vegetables (Rodionova et al. 1995), in water (Baudoin and Davis 1986; Molimard et al. 1995), in insects (Fischer 1999), as airborne spores (Cosentino and Palmas 1991) and even in the human body (Hrdy et al.1995). 簡介 地絲菌型的真菌早在175年以前,就被形容為一種"白色的絲胞屬真菌,菌絲會脫離出 圓柱狀的胞子". 日常生活環境中很容易可以找到它. 地絲菌會在吃的裡頭出現,像是 穀物,起司,酵母,和蔬菜,也可能在空氣中(胞子),水裡,昆蟲,甚至跑到人的身上. Compact disks are used worldwide to store sound, music, images, software, text files, and that they are a secure persistent store is taken for granted. A CD consists of a pregrooved polycarbonate substrate, a metal reflector, an overcoat and often a dye (the latter in CD-R). CD producers (i.e., Philips, Sony) advertise the excellent properties of CDs, describing two threshold temperatures involved with the softening of polycarbonate at 170°C and with a transition of the dye at 265°C (Holtslag et al.1992). There have been few reports describing biodegradation of polycarbonates because they present great chemical stability; in addition, their biocompatibility allows polycarbonates to be used in medical implants (Mathur et al. 1997). Japanese research groups have studied the biodegradability of aliphatic polyestercarbonate (Imada al. 1999) and have isolated proteobacteria (Pseudomonas sp. and Variovorax sp.) from degraded polycarbonates in the river Ibaraki, Japan (Suyama et al.1998). Additional research topics on biodegradability of polycarbonates are their cytotoxicity, water resistance and the ability of fungi to penetrate poly- carbonate micropores, as has been reported by Bardage and Daniel(1997). CD廣泛的用來儲存聲音,音樂,影像,軟體和文字,被視為是安全持久的儲存媒體. CD片是由有溝紋的聚碳酸酯作基底,上面是金屬的反射體,再上層是外殼且通常有染色 (近期的CD-R). CD製造商(如新力,飛利浦)宣傳CD片的優異特性,並說明其兩個門檻 溫度,分別為聚碳酸酯軟化溫度170°C,染色層轉化溫度265°C(註1). 目前已有少數 報告敘述了聚碳酸酯的生物降解,畢竟它們的化學性質實在穩定. 另外,生物上的兼容 性讓聚碳酸酯被拿來製作醫療用植入管. 日本的團隊研究了脂肪族聚酯碳酸酯的生物 可降解性,且從取自日本茨城縣河流中降解的聚碳酸酯上,分離出變形菌. 其他關於 聚碳酸酯生物可降解性的研究,尚有它的細胞毒性,防水性以及真菌對微孔的穿透性 (註2). 註1: 是指熔點Tc跟玻璃轉化溫度Tg?? 似乎不怎麼重要,至於想看CD加熱後會怎樣的, 請點 http://pslc.ws/fire/polyburn/polyburn.htm 註2: ()中的期刊文章就省略不翻,以下同. Compact disk sample Last summer, one of the authors, Victor Cardenes, made a social visit to Belmopan, Cayo District, Belize, Central America, where he stayed with his friends Miguel Cano and Zayda Villar. While talking about life in Belize, Victor's hosts showed him a CD (Fig.1a-c), which contained clearly visible biodeterioration paths on its surface and joked, "Look, life is so strong here, we even have something that eats CDs". The paths on the CD caught Victor's (a geologist) interest because of their similarity with the trails left by burrowing worms, a subject he had recently been studying. Miguel and Zayda gave him the affected CD (by the Kronos Quartet, issued by Elektra Entertainment of Warner Communications, Baltimore,USA) and he brought it back to Spain with him at the end of his trip. At the Spanish Natural Science Museum of the CSIC, Victor decided, with author Dr.Garcia-Guinea, to observe the CD with polarizing and scanning electron microscopes. Together they observed the paths, which formed fractal structures and destruction of the pits and halls in the aluminum on the disk. In search of fungi or bacteria, the disk was taken to the authors, Dr M.J. Martinez and Dr A.T. Martinez, biologists at the Centro de Investigaciones Biologicas (CIB) of the CSIC. Here, under a confocal microscope, the hyphae of a fungus became clearly visible. The CD was cut into two pieces – one for the Museum and the other placed in the CIB in a Petri glass at room tempera- ture and 100% humidity, where the fungus continues to grow. CD試片 去年暑假,作者之一的維克多‧卡登尼斯,到位於中美洲貝里斯,卡右區的貝爾墨邦 訪問,和同行的兩位友人米高‧卡諾和捷達‧維拉在那兒作了停留. 當聊到貝里斯的生活 時,飯店老闆拿了張CD給他看(圖1a–c),並開玩笑說: "瞧,我們這裡東西的生命力特強, 連CD都被當成食物給啃了." 該CD片上有著明顯的生物腐蝕痕跡. 那軌跡興起了卡登尼斯 (身為地質學家)的好奇心,因為看起來很像他才剛剛研究過的,會鑽洞的蠕蟲留下來的 足跡. 米高和捷達把那張受到侵襲的CD(註1)交給卡登尼斯,好讓他在旅途結束時帶回 西班牙. 在西班牙CSIC(註2)所轄的自然科學博物館中,維克多和另一位作者賈西-朱尼亞博士 決定用偏光電子掃描顯微鏡去觀察這張CD. 他們發現那些軌跡呈現不規則碎片形,而碟片 鋁層的資訊槽也遭到破壞(註3). 為了研究到底是真菌還是細菌,片子又轉交給另外兩位 作者安琪拉‧T‧馬丁尼茲和瑪利亞‧耶穌‧馬丁尼茲. 他們是CSIC旗下CIB(註4)的研究 人員. 在雷射掃描共軛焦顯微鏡之下,真菌菌絲清晰可見. 後來CD被切成兩半,一半拿回 博物館,另一半則留在CIB,且置於培養皿中,以室溫/100%相對溼度讓真菌繼續生長. 註1: 文中CD是Kronos絃樂四重奏在Elektra的錄音,但現在廠牌好像變成nonesuch? 因原文語焉不詳,只能推算是2000年以前發行的,無法確定是哪張. 請參考連結 http://kronosquartet.org/records/index.php 註2: Consejo Superior de Investigaciones Cientificas 相當於我們的國科會. 註3: CD上的0跟1好像分別叫作pit跟land,我不曉得文中的"pits and halls"是在說啥. 註4: Centro de Investigaciones Biologicas Results and discussion The study of these paths in the transparent zones of the CD, by transmi- tted and reflected light microscopy, X-ray diffraction and scanning electron microscopy, revealed dendrite microstructures of aluminum–polycarbonate mixtures and the complete loss of the original information pits. Fungal growth and partial degradation of metal and polycarbonate were detected by direct observation of the original CD with a Zeiss photomicroscope coupled to a CCD camera. The hyphae growing in the natural deteriorated areas of the CD were easily observed in the peripheral region under the light microscope (Fig.2a-c). To isolate and identify the fungus, small fragments (2×4 mm) from the deteriorated areas of the Belize CD were superficially sterilized by immersion in 70% ethanol (3 min), washed with sterile water and plated on malt extract (2%) agar and malt extract (2%) + glucose (2%) agar containing cloramphenicol (12 mg/l), and incubated at 26°C in darkness. From the steri- lized CD fragments, a unique type of fungal colony with white aerial mycelium was detected in both media at different incubation times. When the fungus was grown on malt extract agar, white colonies with a velvety surface and a growth rate of 8 mm/day were obtained. Under the light microscope, the fungus dis- played septate hyphae with dichotomous branching. The morphological characte- ristics of the strain isolated from the CD did not change with the presence of glucose in the medium (Allermann et al. 1978). The fungus presents two types of hyphae (2.5-3 and 4-6 μm wide), the main hyphae disarticulating into cylindrical cells (arthroconidia) with 4–6×6–13 μm size. The oldest arthroconidia became barrel-shaped. A difference between arthroconidia from lateral and main branches was not observed. 結果與討論 在研究這片CD透明區域上的痕跡時,共動用了穿透式,反射式顯微鏡,X光繞射儀, 電子掃描顯微鏡,結果呈現出由鋁-聚碳酸酯混雜成的樹突狀的微組織,而原本該有的 資訊槽(pits)則一整個消失. CD上真菌的生成,以及金屬跟聚碳酸酯有部份被降解的 情形,都被接上數位相機的蔡司可照像式顯微鏡給直接捕捉到了. 在光學顯微鏡下可 輕易觀察到,菌絲在CD圓周附近自然變質的區域增生(Fig.2a-c). 為了隔離並確認真菌, 先從這張貝里斯CD的變質部份取下小的碎片(2×4 mm),並做表面消毒,依序為浸放在70% 乙醇(3分鐘),以無菌水清洗,分置於麥芽抽出物(2%)培養基,麥芽抽出物(2%)加葡萄糖 (2%)培養基,內含氯絲菌素(12 mg/l,註1),最後放在26°C的暗室中培養. 各個不同的 孵育時段中,兩份消毒過的CD碎片皆偵測到一種伴隨著白色氣生菌絲,十分獨特的真菌 菌落. 麥芽抽出物培養基這邊,表面光滑的白色菌落生長速度為8毫米/天. 用光學顯微 鏡就可看到有隔膜菌絲,並有岔狀的分支. 對CD上分離出的的本菌種而言,其形貌特徵, 不因葡萄糖的的存在與否而有所不同. 該真菌有兩種菌絲(2.5-3&4-6微米寬),主幹的 菌絲脫出圓柱狀的細胞(桶狀節生胞子),大小為4-6x6-13微米(註2). 最老的桶狀節生 胞子則變成中廣型的桶狀. 而側向跟主分支上的節生胞子,並未發現兩者有什麼不同. 註1: 原文cloramphenicol應是chloramphenicol誤植(?!) 註2: 我看不懂4-6x6-13微米是在說啥,或許6x6是截面積,4~13是長度吧. @@" 事實上後半段翻得還頗語無倫次 +.+ The above characteristics suggested that the fungus isolated from the CD should be classified in the genus Geotrichum. This genus is restricted to ascomycetous species of arthroconidial fungi, whereas the morphologically related genus Trichosporon is reserved for basidiomycetous fungi. The study of carbohydrate composition of fungal cell walls can be used as a taxonomical indicator to differentiate between related fungi with ascomycetous and basi- diomycetous affinities in these and other genera. With this in mind, at the CIB, the mycelium was hydrolyzed (5 M trifluoroacetic acid, 90 min, 120°C) and neutral sugars analyzed as alditol acetates by gas chromatography using a SP-2380 capillary column programmed from 210°C to 240°C, with a 3-min ini- tial hold, a heating rate of 15°C min–1 and a final time of 7 min. The pre- sence of xylose is most often characteristic of basidiomycetous arthroconidial fungi. The absence of this sugar after GC analysis of the cell walls of the fungus isolated from the CD confirmed that it presents ascomicetous characte- ristics. 從上面這些特性看來,CD上的這些真菌應該是歸類在地絲菌屬. 本屬限制為節-分生 胞子的子囊菌種,而其他形態類似的像是絲包菌則分類在擔子菌. 真菌纖維細胞壁的碳水 化合物組成,是區別子囊菌和擔子菌等近似的屬的指標. 基於此理論,她們在CIB先將菌絲 水解(5M三氟醋酸,90分鐘/120°C),藉醣醇乙酸酯分析其中性糖組成,方法是使用SP-2380 毛細層析管,從210~240°C間開始,先加熱3分鐘後,再以每分上升15°C的速度加熱到7 分鐘,作氣相色層分析. 若是節-分生胞子的擔子菌,幾乎都會出現木糖,但此次針對CD片 真菌細胞壁所做的氣相色層分析並未出現,故可確定他呈現出子囊菌的特徵. This agrees with other morphological criteria, such as the absence of blastoconidia. The fungus displays some of the characteristics reported for Geotrichum candidum although the main branches of this fungus are wider. The fact that intense biodeterioration in CDs, destroying the information pits, has been caused by a common fungus (a species close to G. candidum) leads to speculation on the future of compact disks as a secure persistent storage medium for sound, image and computer files. The Belize fungus has been well studied, and thanks to the information explosion and the Internet, we are receiving reports and samples from Guatemala, Mexico, Panama, Costa Rica, Hong Kong and other tropical countries. We now have three pending tasks (1)to determine the geographical and climatical distribution of this bio- deteroration phenomenon, (2) to study the interaction of the fungus with the host materials of the CD and (3) to confirm its systematic relationships by molecular biology techniques. 實驗分析所得,也符合僅依型態標準去判斷的結果,好比說,它並沒有芽生分生胞子. 這真菌呈現出若干已知的白色地絲菌的特點,雖然其主要分支較粗些. 而擺在眼前的,是 CD上由(某種接近白色地絲菌的)真菌引起的嚴重生物腐蝕,且資訊槽不保,這讓人思考CD 是否真為持久可靠的影音/檔案儲存媒體. 貝里斯的真菌已經過研究,拜資訊爆炸和網際 網路盛行之賜,我們又收到許多來自瓜地馬拉,墨西哥,巴拿馬,哥斯大黎加,香港和其他 熱帶國家的樣本與相關回報. 現在,我們仍有三個懸而未決的任務: 1. 針對有這種現象的地區,找出其地域性和天候性的分布. 2. 探討真菌跟其宿主CD材質間的關係. 3. 利用分子生物技術確認其系統性的關連. Acknowledgements We are grateful to A. Prieto for gas chromatography ana- lysis and Angeles Guijarro for technical assistance in Madrid. The work was supported by the project 1FD97–0959-CO3–01 of the Plan Nacional de Inves- tigacion Cientifica y Desarrollo Tecnologico (Spain). Thanks to Josefo Bedoya for his help with the scanning electron microscope. Thanks also to Paul Giblin for critical revision of the manuscript. 我們對A.普利托和安吉莉斯‧古加洛分別在氣相色層分析與技術上給予的幫助表達 由衷感激. 本輯為(西班牙)國家科學技術研究部第1FD97–0959-CO3–01號計畫. 謝謝 喬瑟夫‧比多雅對電子掃描顯微鏡的支援,也同樣的感謝保羅‧吉卜林在原稿修訂上給的 建議. Fig 1 a Deteriorated compact disk from Belize, Central America. b Detail of the bioturbation paths visible to the naked eye. c Scanning-electron microscopy image of a deteriorated fragment of the Belize compact disk showing dendrite textures of the bioturbed aluminum–polycarbonate mixture 圖1 a 中美洲貝里斯,毀損的(被腐蝕的)CD b 生物擾動作用的痕跡用肉眼也可以看得清楚 (300μm可以用肉眼看得清楚??) c 腐蝕的碎片於電子掃描顯微鏡秀出的圖中,有著生物擾動下由鋁和聚碳酸酯混雜成的 樹突狀微組織 Fig 2 a Light microscopy image of the bioturbation paths displaying the fungal growth in the peripheral region of the compact disk alteration. b Hyphae and disarticulated conidia (arthroconidia) in pure culture of the Geotrichum-type fungus isolated from the deteriorated compact disk (light microscopy). c Detail of typical conidial chains produced by this fungus (light micro- scopy) 圖二 a 光學顯微鏡拍到的圖上,生物擾動紀錄痕跡可看出真菌在CD圓週附近變質的區域生長 b 自CD分離出的地絲菌型真菌,做純培養後所得菌絲和脫出的分生子柄(節-分生胞子) c 該真菌特有的分生胞子鏈 待續..... -- 音響的莫非定律 : 每當朋友要來家裡聽音樂時,音響不是莫名的故障,就是衰聲連連. --

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