Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/25624
標題: 接種微生物於重金屬污染土壤對於重金屬移除之可行性評估
Assessment of heavy metals removal from polluted soils by inoculating microorganisms
作者: 楊津銘
Yang, Chin-Ming
關鍵字: 土壤重金屬;Heavy metals;微生物;microorganism
出版社: 土壤環境科學系所
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摘要: 
在台灣及世界各地,許多可耕地都受到重金屬污染。隨著世界人口之增長,需要復育這些污染之可耕地以種植更多之安全農產品,本論文藉由一系列試驗進行以產酸微生物復育重金屬汙染土壤之可行性評估。
浸水土壤系統於土水比1/10 (M/V) 進行試驗,其中包含了3 種處理:僅添加純水、添加含5%蔗糖之溶液以及添加含5%蔗糖溶液並接種產酸酵母菌 (YL, Yarrowia lipolytica) 之處理,每個處理均為3重複進行4個循環,每個循環為期7天。循環步驟如下:1. 秤取5 g土壤於100 mL PE瓶中2. 加入50 mL之去離子水3. 室溫下孵育1週4.以3000 rpm 之離心機離心15分鐘5. 以3M 硝酸酸化抽出液 6.重複步驟2-5 三次。抽出液於分析前經Whatman No.42濾紙過濾,並以AA分析。結果顯示,添加蔗糖後,土壤之pH及Eh 明顯的下降,而金屬包括植物有益元素 (鈣、鎂、鐵以及錳) 以及重金屬 ( 鉻、銅、鎳以及鋅) 均被抽出。重金屬所抽出之濃度與王水抽出之濃度相比,鉻抽出0.03 %,銅抽出0.02 %,鎳抽出14%,鋅抽出90 %之於王水抽出濃度。在第一及第二循環週期,接種YL之處理使得鈣、鎂、鐵以及錳之抽出濃度增加。總體來說,接種YL相較於未接種之處理在金屬鈣、鎂、鐵、錳、鎳以及鉻均有較好之抽出濃度,但於銅以及鋅之抽出效果較未接種之處理差。
在接種真菌之系統當中,土壤水分含量調整至60%之飽和水分含量,試驗方法如下:1. 將5 g 土壤置於 100 mL 之PE瓶中 2. 接種真菌並調整營養及水分至試驗之處理 3. 孵育3或7天以及培養於控制之溫度之下(視試驗所設計而定) 4. 加入25 mL 去離子水並震盪1小時 5. 以3000 rpm 離心15分鐘保留萃取液 6. 再添加50 mL之去離子水並搖晃至土粒散開,將其混合液通過35 mesh 之篩網以保留真菌之菌絲,其上之菌絲以水清洗至乾淨,並於70℃烘箱烘乾後以硝酸 、鹽酸以及過氯酸之混合液分解並以AA分析金屬濃度。萃取液在以AA測定之前以Whatman No.42濾紙過濾。真菌接種之系統中所選用之真菌為Aspergillus niger (An) 以及 887F1 (F),Aspergillus niger為前人依據可溶出骨粉中磷以及金屬之特性所篩選出之真菌;而887F1則是本人由編號887之受多種重金屬污染土壤依據可酸化培養基之特性篩選而得。
結果顯示,接種An之處理對於增加重金屬之移動性有相當大的效果,甚至高於0.1 M HCl抽出;在孵育3天並給予5% 以及10%之蔗糖的處理中,重金屬抽出量來說,鉻抽出80%,銅抽出250%,鎳抽出130%,鋅抽出116%相較於0.1 M HCl之抽出;鉻抽出14%,銅抽出129%,鎳抽出28%,鋅抽出46%相較於王水之抽出。蔗糖添加量5%以及10%之處理並無顯著差異,但對於鉻、銅、鎳以及鉛之抽出濃度來說仍有增加。將孵育時間由3日延長至7日增加了重金屬銅、鉻以及鎳的抽出,但鋅的抽出則是減少的。接種887F1對於重金屬鎳以及鉛的移除與接種An相似,但於重金屬鉻以及銅之移除量相較於An處理少。額外添加氮源使得碳氮比接近10之處理,明顯減少了重金屬鉻、銅、鎳以及鋅之抽出,且pH亦較高,而於菌絲產量上面並無明顯之增加。在溫度控制於20℃ 、28℃以及36℃的試驗當中,887F1於20℃下有較高的菌絲產量,An則於28℃有較高的菌絲產量。培養於真菌培養基在氮濃度固定的狀態下,增加系統中的碳可增加An處理之菌絲產量。

Heavy metals pollution area in arable land has been found in Taiwan, and world-wide. It’s reclamation is a way produce safer agriculture products whose demands are increasing with the world population keeps growing. In this thesis a series of experiments were conducted to assess the possibility to use acid forming microorganisms on the reclamation of heavy metal polluted soils.
In submerged system soil/solution ratio was 1/10 (W/V). There were three treatments, water, 5 % sucrose solution, and 5 % sucrose solution inoculation with acid forming yeast (YL, Yarrowia lipolytica) conducted with 3 replications for four cycles each cycle for 1 week. In this experiment carried out as the following procedures: 1. 5 g of soil put in 100 mL PE bottle, 2. 50 mL of water solution was added, 3. incubated at room temperature for 1 week, 4. centrifuged at 3000 rpm for 15 min, 5. acidifying supernatant with 3 M HNO3, 6. steps 2-5 repeated for three times more. Extracts was filtrated through Whatman No. 42 filter before metals analyzed with AA. The results showed that the addition of sucrose really significantly increased the decrease of soil pH and Eh, and removal of metals, including general plant essential elements ( Ca, Mg, Fe, and Mn ) and heavy metals ( Cr, Cu, Ni, and Zn ). The efficiencies of heavy metal removal was depending on metals, the removed rate based on aqua digestion concentration were around 0.03, 0.02, 14, and 90 % for Cr, Cu, Ni, and Zn, respectively. The inoculation of YL increased the removal of Ca, Mg, Fe, and Mn, especially at the first two cycles of incubation. The inoculation of YL had a better removal than with no inoculation for Ca, Mg, Fe, Mn, Ni, and Cr, but less for Cu, and Zn.
In fungi inoculation system soil water content was adjusted to 60% of saturated water content at the beginning, the procedures were conducted as the following: 1. 5 g soil put in a 100 mL PE bottle, 2. total volume of inoculants and nutrient solutions were made to meet the set water content status, 3. incubated for 3 or 7 days depending on the experimental designs with the incubator temperatures set by experimental designs, 4. 25 mL of deionized water added then shaking for 1 hr, 5. centrifuged at 3000 rpm for 15 min and acidifying the supernatant with 3 M HNO3, 6. 50 mL deionized water was added and shaking for the deposits dispersed then water screened over 35 mesh sieve for collecting the mycelium of fungi. Fungi mycelium was washed with water until clean, then oven dried at 70℃, and digested with HNO3, HCl, HClO4 mixture before metals analyzed with AA. Extracts was filtrated through Whatman No. 42 filter before metals analyzed with AA. Two fungi strains Aspergillus niger (An) and 887F1 (F) were used in this series of experiments, the former was owned by other worker screened for solubilizing phosphate of bone meals, the latter was screened from the No. 887 multi heavy metal polluted soil which also used in this series work.
Results showed that the inoculation of An fungi dramatically increased the removal of heavy metals the removal rate even higher than using 0.1 M HCl extraction at sucrose concentrations at 5 % or 10 % with incubation for 3 days. The removal rates of Cr, Cu, Ni, and Zn were about 80, 250, 130, and 116 % of 0.1 M HCl extraction, respectively, and the corresponding removal rate were about 14 %, 129, 28, 46 % of aqua extraction, respectively. The effect of increase of sucrose concentration from 5 % to 10 % was not significant, although it increased the removal of Cr, Cu, Ni, and Pb. The extension of incubation day from 3 to 7 days increased the removal of Cu, Cr, and Ni, but decreased the removal of Zn. The use of strain 887 F1 showed had a similar potential of removal to An strains on Ni and Pb metals, but lower potential on Cr and Cu metals. The addition of nitrogen to make the C/N ratio of the additive solution at 10 was significantly decreased the removal rates of Cr, Cu, Ni, and Zn, with the higher soil pH, although the weight of mycelium was not significantly affected. The 887F1 strain grows higest at 20 ℃, but the An strain grows higest at 28℃, as them grown in 20, 28, and 36℃conditions with fungi medium. The mycelium growth had been increased of An strain with increasing sucrose concentration at constant nitrogen concentration.
URI: http://hdl.handle.net/11455/25624
其他識別: U0005-2808201309281500
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