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年度	108	專案性質	實驗性質
專案類別	研究專案	研究主題	整治
申請機構	國立高雄科技大學	申請系所	海洋生物技術系
專案主持人	鄭至玉	職等/職稱	副教授
專案中文名稱	利用白腐真菌胞外酵素淨化戴奧辛污土之技術研發
中文關鍵字	白腐真菌, 酵素, 戴奧辛, 生物處理法
專案英文名稱	Investigation of extracellular enzyme of white-rot fungi for the bioremediation of Dioxin-contaminated soil
英文關鍵字	bioremediation, Dioxin-contaminated soil, extracellular enzyme,white-rot fungi
執行金額	700,000元
執行期間	2019/1/1 至 2019/12/31
計畫中文摘要	無論從環境、生態、經濟和社群等各方面綜合觀點來看，運用生化科技整治污染，儼然是綠色且永續導向型（green sustainable remediation, 簡稱GSR）整治的一種創新典範。由於具強分解木質素能力的白腐真菌對戴奧辛類的持久性有機污染物具有較高分解活性，因此，本計畫主要探討以台灣本土白腐真菌之胞外複合酵素淨化戴奧辛污土的可行性。 台灣本土95件樣品的田野調查分析結果顯示，真菌具有的漆酶活性顯著高於動物與植物，而8件市售食用菇的活性，則以美人豪菇最高，經ITS基因比對初步鑑定菌種為Pleurotus eryngii。為了降低生產成本，改以農業廢棄資材取代培養基培養白腐真菌Phanerochaete chrysosporium的研究結果顯示，5種再生資材包括木屑、米糠、紅茶渣、綠茶渣與咖啡渣除成功降低酵素生產成本之外，以綠茶渣、紅茶渣與咖啡渣所生產的酵素與控制組PDB相比較，活性增加約1.5, 2.4 and 2.3倍。此胞外複合酵素活性在pH 5與6有最高過氧化氫酶與去除自由基能力。田口實驗發現，適當有機酸的添加貢獻度高達66%，而溫度(25-35℃)、1%以內的介面活性劑(Tween、Triton)則較無明顯活性變化，其中丙二酸與蘋果酸的添加可增強酵素活性約20%。 將未經滅菌、乾燥的污染土壤(PCDD / Fs濃度為7100-8000 ng I-TEQ /kg（或約3-4.75 mg / g）直接與Phanerochaete chrysosporium生產的胞外複合酵素以1：1 混合，並在室溫（約26±2℃）下進行淨化反應96小時。結果發現，各式酵素平均I-TEQ去除率可達49%±6%，其中以咖啡渣再生資材培養6天的胞外複合酵素對戴奧辛I-TEQ可達 60％，總量去除率高達91%。由於反應前後，OCDF與OCDD等明顯下降，但17種戴奧辛均無明顯增加趨勢，因此初步判定此法是以非專一性的開環方式進行降解，屏除了會有更毒戴奧辛產生的可能性。 分析反應12-96小時之間的戴奧辛變化發現，此防治方式在前12小時已完成90%的淨化反應，暗示了再縮短反應時間的可能性。此外，運用木質素過氧化酶（LiP）與錳過氧化酶（MnP）等的介質(mediators)調節特性，添加了一新型介質¬¬¬--硼酸果糖複合體，則又可再一次提高降解效率約17%，目前業已著手申請專利中。 這提供了以後更易復育戴奧辛污土，並兼顧節省能耗、減排空污、節省用水與減免廢水、循環使用綠色原物料資材與減廢、提升土地肥力及豐厚生態多樣性等GSR整治，期能儘速實現淨化戴奧辛污土的最佳管理實務。
計畫英文摘要	As regards to environmental, ecological and social aspects, the use of biochemical technology to remediate dioxin (PCDD/Fs) pollution is more green and sustainable remediation (GSR). White rot fungus degrade the complex and recalcitrant polymer lignin by their ligninolytic enzymes. These enzymes are versatile, extracellular and also able to oxidize various environmental pollutants. Herein, this project mainly explores the bioremediation of the indigenous white rot fungus in Taiwan and its extracellular multiple enzymes to dioxin-contaminated soil. The field survey of 95 samples from Taiwan showed that the fungi had significantly higher laccase activity than animals and plants. The highest activity of 8 commercially available edible mushrooms is in “the beauty of the oyster mushroom”, and the preliminary identification of the strain by ITS gene is Pleurotus eryngii. In order to reduce the production cost, five renewable agricultural waste materials including wood chips, rice bran, black tea residue, green tea residue and coffee grounds was used to cultivate the white rot fungus Phanerochaete chrysosporium. The extracellular multi-enzyme was obtained from the supernatant of 6-day cultivation by five renewable materials. When compared with PDB medium, the total enzyme activity (MnP+LiP) of extracellular multi-enzyme produced by green tea residue, black tea residue and coffee grounds were increased to 1.5, 2.4 and 2.3 times. The optimum pH were pH 5 and 6 for peroxidase and free radical scavenging activities, respectively. Malonic acid and malic acid can enhance the enzyme activity by about 20%; conversely, temperature (25-35oC) and surfactant (Tween 80 or Triton X-100, less than 1%) have no significant influence. After 6-day of liquid cultivation, extracellular multi-enzyme obtained by centrifugation were used to examine their enzyme activities and to treat the same weight of non-sterilized contaminated soil under 26±2°C for 96 hours. In comparison with a normal medium (PDB), comparable removal efficiencies by extracellular multi-enzyme cultured with agro-industrial residues were found and the average efficiencies were approximately 49%±6% for P.C. The highest PCDD/Fs removal efficiency reached 60% by TEQ-concentration unit and 91% by mass-concentration unit by extracellular multi-enzyme cultured with coffee ground. Besides, OCDF and OCDD were significant decreased and there is no corresponding increasing sign of other dechlorinated dioxins or furans. This results suggest that PCDD/Fs removal mechanism is biased towards oxidation rather than dehalogenative reduction. Besides, the removal efficiency 90% was achieved within the first 12 hours and was increased by 17% by adding borate-fructose complex as mediator. At present, the patent has been applied. These provides a promising way to regenerate Dioxin soil more easily and to be a good GSR remediation satisfied the best management practice such as saving energy, reducing air pollution, saving water, recycling green raw materials and reducing waste, improving soil fertility and enriching ecological diversity.