標題: | 以硫酸還原菌進行生物沉澱重金屬銅離子之研究 Bioprecipitation of Copper Ion Using Sulfate-Reducing Bacteria |
作者: | 徐秀鳳 Hsu, Hsiu-Feng 林志高 Lin, Jih-Gaw 環境工程系所 |
關鍵字: | 硫酸還原菌;聚乙烯醇;重金屬;螢光原位雜交;中央合成設計法;反應曲面法;sulfate reducing bactera (SRB);polyvinyl alcohol (PVA);heavy metal;fluorescence in-situ hybridization (FISH);central composite design (CCD);response surface Methodology (RSM) |
公開日期: | 2009 |
摘要: | 硫酸還原菌 (sulfate-reducing bacteria, SRB) 可藉由生物沈澱過程產生不溶性金屬硫化物,具有極高的生物復育潛能,因此近年來專家學者積極發展此技術去除廢水中之有毒重金屬。本研究以分子生物方法包括螢光原位雜交法 (Fluorescence in situ hybridization method) 及巢式聚合酶鏈-變性梯度電泳法,分析經馴養之硫酸還原菌之菌群結構。分析族群結構顯示,碳針EUB338 佔 DAPI 百分比約為 52%,專一性的探針 (family probe, SRB385及SRB385db) 雜交結果顯示硫酸還原菌佔 DAPI 百分比約 42%,主要以 Desulfosarcina, Desulfofaba, Desulfococcus, Desulfomusa, Desulfonema and Desulfofrigus sp. 等菌為主 (genus probe, DSS658)。
萃取之 DNA 片段經巢式聚合酶鏈-變性梯度電泳法分離,定序後之 DNA序列經由 NCBI GenBank 16S rDNA 資料庫序列比對,結果顯示馴養槽之優勢族群屬於 δ-Proteobacteria,主要歸類為 Desulfovibrio 之相近菌群,Desulfovibrio vulgaris 與 Desulfovibrio vulgaris subsp. vulgaris 其相似度達99%。加入重金屬銅後 16S rDNA 優勢族群仍為 δ-Proteobacteria 之 Desulfovibrio 相近菌群。 Desulfovibrio 屬向來被視為是硫酸還原作用最重要的一群菌群。
利用分批式試驗及掃描式電子顯微鏡/能量分散式光譜儀分析沉降物之元素組成,驗證生物沉澱技術中重金屬離子去除之機制。硫酸鹽及重金屬銅離子初始濃度分別為 300 及 10.0 mg/L。結果顯示未添加 SRB mixed culture兩試驗組均顯示無硫酸還原作用產生,以推測銅離子之去除以化學沈澱作用為主,有無添加碳源組其重金屬銅離子去除率分別為 36 及 44%。添加 SRB mixed culture 未添加碳源試驗組結果顯示亦無硫酸還原作用發生,推測重金屬銅離子之去除主要來自SRB mixed culture之吸附作用,其 Cu+2 去除率約 40%。添加 SRB mixed culture、碳源及 Cu+2 實驗組之硫酸還原率達 98%, Cu+2 去除率達 99.2%,SEM/EDS分析結果S元素產生來自硫酸還原菌之作用,推測藉硫離子產生硫化銅 (CuS) 之生物沈澱作用之 Cu+2 去除率約 31%。
研究顯示生物沉殿系統應用受限之主要原因有二:(ㄧ) 硫酸還原菌為厭氧菌,生長速率相對緩慢,致使於連續操作程序硫酸還原菌易因washout作用,使硫酸還原效率降低;(二) 重金屬離子對硫酸還原菌大都具有毒性,抑制硫酸還原作用使效率降低。為改善連續系統厭氧菌生長速率緩慢及重金屬離子之毒性作用,本研究應用聚乙烯醇 (polyvinyl alcohol) 固定SRB mixed culture之可行性,並與懸浮生長SRB mixed culture比較,於含10.0及23.2 mg/L銅離子濃度系統之硫酸還原率及銅離子去除率之差異。實驗結果顯示懸浮生長SRB mixed culture試驗組於銅離子初始濃度10.0 mg/L系統中生長正常,且硫酸鹽還原率於第2 d達93%,銅離子去除率達99.2%;於銅離子初始濃度23.2 mg/L系統之SRB 還原作用,則因銅離子毒性之影響明顯受抑制,於第7 d其硫酸鹽還原率僅14.4%,且銅離子去除率僅40.7%。聚乙烯醇固定SRB 擔體試驗組之結果顯示其銅離子去除率於第2 d即達99.2%,硫酸鹽還原率亦達98%,聚乙烯醇固定 SRB擔體之硫酸還原作用正常,未被抑制,明顯與懸浮生長 SRB mixed culture系統有差異,顯示聚乙烯醇固定 SRB 擔體試驗組之硫酸還原作用未被抑制,推測因聚乙烯醇包覆 SRB mixed culture,降低銅離子對 SRB 毒性,使生物沉殿更有效應用於以處理重金屬銅離子廢水。
藉由中央合成設計法 (Central Composite Designs,簡稱 CCD)設計11組試驗,操作因子為重金屬銅離子濃度及聚乙烯醇固定 SRB 擔體添加量對硫酸還原作用影響進行探討,並搭配反應曲面法 (Response Surface Method,簡稱 RSM),分析找出最佳的評估指標及最佳的操作條件。實驗結果顯示各組硫酸鹽濃度在第168小時的總去除率皆在99%以上,而在第168小時的銅離子濃度皆小於 1 mg/L,但明顯看出最高銅離子濃度 (Run7) 及最低PVA 固定SRB 擔體添加量 (Run4) 兩組試驗有明顯遲滯期。分析結果顯示,選擇硫酸鹽還原之反應速率常數為主要評估指標,反應曲面呈收斂之趨勢,有最佳值的存在,SRB mixed culture量則為138.5 mg VSS/L,而銅離子初始濃度在 51.5 mg/L時,反應溫度控制在30±2℃時,有最高硫酸鹽還原之反應速率常數為 1.57 1/d。
藉由掃描式電子顯微鏡/能量分散式光譜儀分析聚乙烯醇生物擔體內沉降物質之元素組成,空白聚乙烯醇擔體控制組因無硫酸還原作用發生,分析結果亦顯示均無硫 (S) 元素存在;聚乙烯醇固定 SRB 擔體組均有明顯比例之 S 元素存在。除此之外含有 Ca、 Fe 及 P 元素,均為培養基之成分。亦驗證 S 的產生主要來自添加 SRB mixed culture 產生硫酸還原作用所產生,顯示生物沉澱去除眾金屬之機制主要來自硫化金屬物質之產生。 A sulfate-reducing bacterial mixed culture was successfully enriched from seed sludge of anaerobic sludge digester of a swine wastewater treatment plant. The population of sulfate-reducing bacteria (SRB) in the enriched culture (around 42%) was characterized by fluorescence in-situ hybridization with group and genus specific 16S rRNA-targeted oligonucleotide probes. Desulfosarcina, Desulfofaba, Desulfococcus, Desulfomusa, Desulfonema and Desulfofrigus sp. were identified by probe DSS658 as the dominant species of the enriched SRB mixed culture. Subsequently, batch experiments were conducted at copper ion and sulfate concentrations of 10.0 mg/L and 300 mg/L, respectively, to quantify the ability of the enriched SRB mixed culture in simultaneous sulfate reduction and Cu+2 removal. Sulfate reduction efficiencies of the culture in the absence (biotic system without Cu+2, BS-1) and presence (biotic system with Cu+2, BS-2) of Cu+2 were 96.8% and 98.8%, respectively, after 6 d. In BS-2, 99.2% Cu+2 removal was observed after 1 d. However, 67% of copper ion was removed by chemical precipitation and bioaccumulation. No significant inhibition of bacterial growth was observed at the Cu+2 concentration studies, i.e. 10.0 mg/L. The chemical precipitation as hydroxide/carbonate caused a copper ion removal of 44% in AS-1 (abiotic system without lactate) and 36% in AS-2 (abiotic system with lactate), after 6 d. And, the majority of copper ion was removed as copper sulfide well before the occurrence of copper hydroxide precipitation. The energy dispersive X-ray spectroscopy analysis of the precipitates obtained from biotic and abiotic systems confirmed the origin of copper sulfide as the result of SRB mixed culture. In order to apply the enriched SRB mixed culture for copper ion removal at higher concentration, the mixed culture was immobilized in polyvinyl alcohol. Subsequently, the effect of using a SRB mixed culture immobilized in polyvinyl alcohol for the simultaneous sulfate reduction and Cu+2 removal was investigated. Batch experiments were designed using central composite design with two parameters, i.e. Cu+2 concentration (10-100 mg/L) and the quantity of immobilized SRB mixed culture (19-235 mg of VSS/L). The response surface methodology was used to model the experimental data and to identify the optimal conditions for the maximum sulfate reduction and Cu+2 removal. In the optimum condition, i.e. ~138.5 mg of VSS/L of SRB mixed culture immobilized in polyvinyl alcohol, and ~51.5 mg/L of Cu+2 concentration, the maximum sulfate reduction rate of 1.57 1/d was observed using the first-order kinetic equation. Overall, immobilizing the sulfate-reducing bacterial mixed culture in polyvinyl alcohol can enhance the Cu+2 removal and the resistivity of the culture towards copper ion toxicity. Also the enriched SRB mixed culture could be applied for bioprecipitation Cu+2 removal at elevated concentrations. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT079019807 http://hdl.handle.net/11536/40259 |
顯示於類別: | 畢業論文 |