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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17422

Title: 硫酸還原菌與鐵還原菌生理生化鑑定及其生物復育之應用研究
Isolation & characterization of some sulfate-reducing & iron-reducing bacteria, and application of these bacteria in bioremediation
Authors: Chih-Hung Chen
陳智宏
Contributors: NTOU:Institute of Marine Biology
國立臺灣海洋大學:海洋生物研究所
Keywords: 鐵還原菌;硫酸還原菌;五氯酚;脫色;偶氮染劑;三苯甲烷染劑;蔥醌染劑;生物分解
Shewanella;iron reducing bacteria;sulfate reducing bacteria;decolorization;pentachlorophenol;azo dye;triphenylmethane dye;anthraquinone dye;biodegradation;biocorrosion
Date: 2008
Issue Date: 2011-07-04
Abstract: 過去諸多的研究報告指出,鐵還原菌以及硫酸還原菌等厭氧微生物在環境污染物生物復育過程中佔有相當重要的角色與地位,因此本論文希望藉由化學分析技術、分子生物技術以及純種微生物培養及鑑定技術,探討鐵還原菌以及硫酸還原菌在於環境污染物如五氯酚、環境金屬生物腐蝕過程或是染料成分分解應用時之應用價值。 首先在環境污染物五氯酚方面,作者過去的研究發現,淡水河關渡宮河口底泥內的厭氧微生物族群具有五氯酚還原脫氯的能力,而且不同電子提供者或電子接受者加入底泥中,會造成五氯酚不同還原脫氯的速率,因此在本研究中欲以添加不同電子提供者(乳酸鹽或丙酮酸鹽)與電子接受者(硫酸鹽或亞硫酸鹽)於河口底泥中,探討在不同基質利用狀態下五氯酚脫氯速率以及底泥中主要的脫氯菌群為何。結果發現,添加乳酸鹽與硫酸鹽培養時五氯酚之脫氯速率為其他組的8倍以上,同時五氯酚脫氯伴隨硫酸還原作用而進行,非但不會抑制五氯酚脫氯,反而明顯的促進脫氯菌群進行五氯酚脫氯,顯示脫氯菌不受硫酸還原作用抑制,而就前人相關的研究中得知,厭氧河口或海洋環境中分離出的脫氯菌大都是硫酸還原菌屬之Desulfovibrio。因此本實驗中以Desulfuromonas, Desulfitobacterium, Dehalococcoides, Desulfomonile teidjei, Desulfotomaculum, and Desulfovibrio專一性的引子作為探討五氯酚脫氯過程中脫氯菌種類確認的證據之一,在各五氯酚脫氯實驗組萃取所需之DNA,再以專一性引子經PCR放大後發現,所有實驗組無論是甲烷生成狀態(methanogenic condition)、硫酸還原狀態(sulfate reducing condition)、亞硫酸還原狀態(sulfite reducing condition)或是丙酮酸發酵狀態(pyruvate fermentation condition)下,即使經過巢式聚合酶鏈反應(nested PCR),所有基因序列(gene sequence)中完全沒有發現任何Desulfomonile或Desulfotomaculum的基因序列,其他基因序列除Desulfovibrio以外,包括Desulfuromonas, Desulfitobacterium, Dehalococcoides雖存在於大部分的底泥中,但培養時額外添加五氯酚並不能誘導這些菌群數量增加,而且他們的基因序列個別會受到硫酸鹽或亞硫酸鹽、亞硫酸鹽、五氯酚之影響而無法像無添加培養之底泥中穩定存在,甚至菌株Dehalococcoides與Desulfitobacterium的基因序列會因為硫酸鹽添加之後的硫酸還原狀態而減少,反之Desulfovibrio大量存在於各底泥中,且數量遠高於其他三個脫氯菌群,更在硫酸還原脫氯狀態下旺盛的生長,所以認為Desulfovibrio應該是此河口底泥在五氯酚脫氯過程中優勢的菌群,並且也推測菌株Desulfovibrio的存在會與五氯酚的脫氯有很大的關連性。Desulfovibrio之基因序列經變性梯度膠體電泳(DGGE)分離後顯示,添加乳酸鹽與硫酸鹽或亞硫酸鹽培養後相較於其他組別額外多出二個明顯的亮帶,共有六個亮帶,也由於硫酸還原狀態下五氯酚的脫氯速率明顯增加,而且經變性梯度膠體電泳(DGGE)表較之後菌群種類有顯著著差異,所以本實驗更進一步針對硫酸還原狀態下的基因進行基因選殖(gene cloning),結果證Desulfovibrio菌群中包含有兩株菌株的基因序列分別與Desulfovibrio strain TBP1以及Desulfovibrio strain PCP1最接近, 而這兩株菌株過去有都主要是應用於氯酚類化合物或溴酚類化合物之生物降解之用,結合所有證據包括五氯酚降解速率結果、PCR基因放大實驗、變性梯度膠體電泳、基因選殖以及過去碩士班以16S rRNA探針標定的實驗,有充分的證據證明Desulfovibrio菌屬為淡水河關渡宮底泥中降解五氯酚主要之硫酸還原菌族群。 在生物腐蝕研究方面,本實驗採取中油熱交換冷卻水系統腐蝕管線沈積物,以分子生物鑑定、最大可能數(MPN)微生物計數方式、微生物純化培養、金屬腐蝕率計算、電子顯微鏡觀察以及元素分析方式,希望瞭解硫酸還原菌和其他菌株在金屬管線腐蝕過程中扮演的角色,期望瞭解中油熱交換冷卻水系統腐蝕管線沈積物中分離出的菌種,包括:硫酸還原菌Desulfovibrio、Shewanella以及Clostridium,三者在中油金屬管線腐蝕過程所扮演的角色,實驗結果發現整個冷卻水系統各管線水樣中都只有少量的硫酸還原菌存在,而且只有少部分被腐蝕的金屬沈積物呈現黑色與硫酸還原菌進行硫酸還原作用產生之硫化鐵,其中大多被腐蝕的金屬沈積物是呈現黃色至深褐色的顏色,對整個研究結果所代表的意義都釐清之後,認為冷卻水循環系統中除少部分金屬生物腐蝕作用為厭氧硫酸還原菌所主導外,另外應該還有更重要的微生物族群主導冷卻循環水系統的生物腐蝕作用,這些微生物族群可能包含一些鐵還原菌(如:Shewanella或Geobacter)以及可提供金屬腐蝕氧化還原過程電子的產氫菌, 首先我們選用一組專為硫酸還原酵素(sulfite reductase)設計的引子(primer)DSR1F與DSR4R,測試菌株與硫酸還原的關連性,結果菌株中只有Desulfovibrio sp.與Shewanella sp.具有硫酸還原酵素(sulfite reductase),後續的研究也證實這兩株菌都可以利用硫酸鹽並產生更還原態之其他硫化物,例如:亞硫酸鹽等,因此也認為,如果中油冷卻循環水系統之生物腐蝕與硫酸鹽之還原有關,那Desulfovibrio sp.與Shewanella sp.同時都有參與反應的機會就相當高。而另外一個實驗發現我們所分離的菌株Clostridium sp.具有產氫之能力,氫氣產能大約佔血清瓶中上層氣體12.6 ~ 21.2%(大約可以產生6.3 ~ 10.6 ml以上體積之純氫氣),然而在我們的研究中也發現菌株Shewanella sp.可以有效利用氫氣作為電子提供者,因此在生物腐蝕作用中,菌株Clostridium sp. 與Shewanella sp的關連性就顯而易見了。 在實驗室層級,以單一菌株Clostridium sp.、Desulfovibrio sp.和Shewanella sp.、或菌株與菌株兩兩混合、或三株菌珠混合培養之後對碳鋼片進行生物腐蝕,實驗中無論是菌株生物量或生長情形(OD600)、pH值的變化或是碳鋼腐蝕速率(miles per year; mpy)之計算,都證實當三株菌株混合時隨時間變化,總菌量明顯增加、pH值顯著下降而碳鋼片腐蝕速率也大大提升。若單獨以菌種差異來看,Desulfovibrio sp.之碳鋼腐蝕能力最強約為0.184 mpy (miles per year),其次為Clostridium sp.的0.034 mpy,最差為Shewanella sp.的0.019 mpy,當兩種純菌混合測試時發現,與Desulfovibrio sp.混合的組別腐蝕效率會高些,如:Desulfovibrio sp.和Clostridium sp.混合時腐蝕速率為0.38 mpy,Desulfovibrio sp.和Shewanella sp.混合時腐蝕速率為更高的0.44 mpy,而Shewanella sp.和Clostridium sp.混合之腐蝕速率就差了許多為0.23 mpy,不過腐蝕情況最嚴重的還是當三種菌株混合時,腐蝕速率可高達0.84 mpy,相較於Desulfovibrio sp.單一菌株之腐蝕速率為0.184 mpy,明顯快上4.5倍,這樣的結果也相對證實了中油冷卻循環系統碳鋼金屬腐蝕的時候,並非全靠一株硫酸還原菌Desulfovibrio sp.菌株,當有其他菌株例如本實驗中的Shewanella sp.和Clostridium sp.參與反應時,腐蝕的情況可能以數倍的方式成長,但不可否認的是,單就單一菌株或菌株兩兩混合時之腐蝕速率來看,硫酸還原菌還是金屬腐蝕的主要元凶,此外若是配合OD600與pH數值變化來看,在培養基中加入鐵片作為電子接受者時,結果能有效提升菌株或菌群之OD600值,而混合菌菌量多寡也相對應於腐蝕速率的變化,在OD600值的結果為:Clostridium+Dsulfovibrio+Shewanella > Desulfovibrio+Clostridium > Desulfovibrio+Shewanella > Clostridium+Shewanella > Desulfovibrio,金屬腐蝕速率結果相同也是類似於OD600值的結果,因此認為生物量(biomass)與其生物腐蝕(biocorrosion)有較大的關連性,而pH變化不會因為培養基中多加鐵片使pH降至更低,不過pH變化似乎與金屬腐蝕上也有相對應之關連性的關連性,因此認為pH變化也有可能影響金屬之生物腐蝕作用,但是因為數據資料有限,所以在此仍然對pH與生物腐蝕相關性保持保留態度。 由煉油廠冷卻系統中分離出的兼性厭氧鐵還原菌株,經進一步鑑定之後確認為Shewanella菌屬,同時也命名為Shewanella decolorationis NTOU1,其鑑定結果如以下所示:此革蘭氏陰性菌株屬於γ proteobacteria,沒有孢子產生,有單一極性鞭毛(single polar flagellum),培養於培養基上菌落外觀呈現圓形不透明橘色,橘色色素其吸光值在342 nm與396 nm具有高峰,菌體大小約為0.70~0.85 μm(寬)×1.5~2.5 μm(長),可生長在pH5~pH9之間、鹽度0‰~75‰之間、溫度10~40 oC,生化特性測試菌株具有觸酶(catalase)、氧化酶(oxidase)、澱粉酶(amylase)、尿素酶(urease)、明膠酵素(gelatinase)以及硫酸還原酵素(sulfite reductase)活性,而不具有脂肪酶(lipase)活性,可利用Fe(III)、Mn(IV)、nitrate、iron oxide、sulfate、sulfite、thiosulfate、sulfur、arsenate以及selenate作為厭氧呼吸作用之電子接受者,以H2、lactate、pyruvate或formate作為生長所需之電子提供者,細胞含有Coenzyme Q =泛醌(Ubiquinone):主要為Q7與Q8,細胞膜上之主要脂肪酸組成為C16:0、 iso-15:0、C16:1ω7c以及17:1ω8c,沒有發現20:5ω3的存在,DNA序列之G+C比例為50.3 mol %,基於1150 bp的16S rDNA基因序列片段放大分析,並與另外21株Shewanella菌種比對發現,菌株NTOU1之16S rDNA基因序列分別與S. decolorationis 以及S. putrefaciens有97%以及96%的相似度,另外再以1040 bp的gyrB基因序列鑑定發現,分別與S. decolorationis 以及S. oneidensis有97%以及90%的相似度,與S. decolorationisDNA-DNA雜交的測試實驗分析結果為74.4%,根據以上分子生物方法分析結果,都顯示此菌株應同屬於S. decolorationis,因此將菌株命名為S. decolorationis NTOU1,目前保存於台灣新竹食工所生物資源保存及研究中心(Bioresource Collection and Research Center; BCRC)編號為BCRC 910321T以及日本菌種保存中心(Japan Collection of Microorganisms; JCM)編號為JCM 14211T。 也因菌株S. decolorationis具有染劑脫色能力,所以在本研究中我們測試菌株S. decolorationis NTOU1是否也具脫色降解偶氮染劑(azo dye)、三苯甲烷染劑(triphenylmethane dye)及蔥醌染劑(anthraquinone dye)的能力,首先針對偶氮染劑研究之結果發現,菌株S. decolorationis NTOU1可在沒有電子載體(electron carrier)的狀況下有效的將多種偶氮染劑﹝剛果紅(congo red)、蘇丹黑(sudan black)、酸性橙(acid orange)、橘G (orange G)、甲基橙(methyl orange)及甲基紅(methyl red)﹞脫色降解,而脫色降解過程最適的酸鹼度值及溫度分別為pH 6.0-7.0 及 30oC-40oC。實驗中發現以氫氣(H2)、甲酸鹽( formate)及丙酮酸( pyruvate)為電子提供者時有最好的脫色效率,添加檸檬酸鐵(ferric citrate)、鐵氧化物(manganese oxide)、硒酸(selenate)、鐵氧化物(ferric oxide)、硝酸(nitrate)或硫酸(thiosulfate)時可增加菌株S. decolorationis NTOU1對剛果紅之脫色率。在剛果紅厭氧脫色完成後轉至耗氧條件下培養,剛果紅之厭氧脫色產物,聯苯胺(benzidine),可在耗氧狀態下由菌株S. decolorationis NTOU1更迅速的進一步分解,這樣的結果證實菌株S. decolorationis NTOU1不僅可以有效的處理有色之染劑廢水,更可以在後續耗氧狀態下進一步處理高毒性之苯胺類代謝產物。 菌株S. decolorationis NTOU1在厭氧鐵還原條件下,以20 mM甲酸鹽(formate)作為電子提供者(electron donor)可以迅速降解多種的三苯甲烷染劑(basic fuchsin, bromophenol blue, crystal violet, malachite green, and methyl violet B),以200 mg l-1濃度條件下三苯甲烷染劑分解速率快慢依序為:malachite green > crystal violet > methyl violet B > basic fuchsin > bromophenol blue,菌株S. decolorationis NTOU1不僅可廣泛的分解多種三苯甲烷染劑,同時也可以對高濃度的三苯甲烷進行分解,以結晶紫為例,即使結晶紫染劑濃度高達1500 mg l-1時菌株S. decolorationis NTOU1仍可有效的將結晶紫脫色(315.7 mg l-1 h-1)並去除毒性,其脫色之最適pH值及溫度分別為pH 8-9 及30-40oC。 在實驗中也發現菌株S. decolorationis NTOU1對結晶紫脫色過程中,如果添加檸檬酸鐵(ferric citrate)非但不會抑制結晶紫的脫色,相反的可以促進結晶紫的脫色,相較於檸檬酸鐵若是添加其他電子接受者,如:硫代硫酸鹽(thiosulfate)、三價氧化鐵(ferric oxide)或氧化錳(manganese oxide)脫色速率則會有些微的下降,此外若以亞硝酸鹽(nitrite) (20 mM)作為脫色時之電子接受者,則菌株S. decolorationis NTOU1的脫色能力會被完全抑制,根據GC/MS偵測結晶紫經此菌株脫色前後之產物可看出,結晶紫會先被還原裂解為Michler’s ketone及N,N-dimethylaminophenol。Michler’s ketone 又可進一步裂解N,N-dimethylaminobenzaldehyde及 N,N-dimethylaminophenol。Michler’s ketone 也可去甲基化而成 [4,4’-dimethylamino phenyl] [4-methylaminophenyl] benzophenone。根據GC/MS偵測孔雀綠經菌株S. decolorationis NTOU1脫色前後之產物可看出,孔雀綠經脫色後之中間代謝產物主要包括有leucomalachite green、 N,N,N’-trimethyl-4,4’-benzylidenedianiline、[N,N-dimethylaminophenyl] [phenyl] benzophenone與N,N-dimethylaminophenol,根據GC/MS偵測甲基紫經菌株S. decolorationis NTOU1脫色前後之產物可看出,甲基紫經脫色後之中間代謝產物主要包括有leucomethyl violet B、N,N’-bis [dimethylamino] benzophenone (Michler’s ketone)、[N,N-dimethylaminophenyl] [N’-methylaminophenyl] benzophenone、N,N-dimethylaminobenzaldehyde、N,N-dimethylaminophenol、 N-methylaminobenzaldehyde與N-methylaminophenol。此外我們還分別利用老鼠組織細胞clone L-929以及菌株E. coli strain JM 109,針對結晶紫、孔雀綠以及甲基紫三種三苯甲烷染劑在脫色前後的細胞毒性性或抑制微生物生長毒性進行測試,也發現三種測試的三苯甲烷染劑經菌株S. decolorationis NTOU1脫色後之產物毒性有非常明顯的下降,顯示菌株S. decolorationis NTOU1脫色前後對三苯甲烷染劑的脫色過程不僅止於脫色作用,同時也是相當好的一個去毒步驟,這樣的結果說明了,菌株S. decolorationis NTOU1除了利用氧化還原的方式將多種三苯甲烷染劑,如:結晶紫、孔雀綠或甲基紫等染劑的顏色去除之外,還可以將這些三苯甲烷染劑的結構進一步還原降解成結構更簡單的化合物,本實驗的圖表之中也清楚呈現菌株S. decolorationis NTOU1降解三苯甲烷染劑的降解途徑,或降解過程中中間代謝產物相對應時間的關係圖,染劑代謝產物毒性測試的實驗更發現,菌株S. decolorationis NTOU1除了可以去除染劑顏色,更可以去除染劑原有之細胞或抑制微生物之毒性,這樣完整瞭解菌株S. decolorationis NTOU1對三苯甲烷染劑最佳的的脫色降解條件、脫色能力的鑑定、對整個染劑脫色降解時化學結構之變化途徑的鑑定以及脫色過程相對應之去毒效果,證實了菌株S. decolorationis NTOU1未來應用於三苯甲烷染料廢水處理之可行與潛力性,也相信這些數據對於未來應用菌株S. decolorationis NTOU1於三苯甲烷染劑廢水生物處理,應該會有相當大的助益。 除了偶氮染劑與三苯甲烷染劑之外,菌株S. decolorationis NTOU1還可以對多種蔥醌染劑包括RB4、RB19、MR11、DR15與DB3進行生物降解脫色,菌株脫色最適合之酸鹼值及溫度條件分別為pH 8.0-9.0以及45oC,脫色過程中添加電子提供者formate以及電子接受者ferric citrate可以達到最佳之脫色速率,而初始脫色速率會隨著初始添加的染劑濃度(100 mg l 1 ~ 1000 mg l-1)增加而增加,當初始脫色速率增加時,染劑脫色百分比大多也會相對提升,以GC/MS分析蔥醌染劑RB4與RB19脫色後的代謝產物,測得包括:1-amino-anthraquinone、2,3-dihydro-9,10-dihydroxy-1,4-anthracenedione與leuco-1,4-diaminoanthraquinone,而MR11代謝產物中測得1-hydroxy-9,10-anthracenedione, DR15代謝產物中測得1,4-dihydroxy-9,10- -anthracenedione and 2,3-dihydro-9,10-dihydroxy-1,4-anthracenedione,DB3代謝產物中測得leuco-1,4-diaminoanthraquinone。 在本研究中發現無論是硫酸還原菌或是鐵還原菌,常常可以在多樣化的環境中發現,除此之外也經常與環境污染物的生物降解息息相關,也由於兩種菌彼此間生存之氧化還原電位不同、可以利用之基質不同或是酵素系統上的差異,使得兩種菌之間可能存在電子交換的可能性,這樣的可能性也環境污染物之生物復育帶來更多的潛力,因為環境中存在的微生物種數以千萬計,所以環境透過微生物降解污染物的自淨能力是可以被期待的,綜合來說,事實上過去在自然環境的污染物生物復育研究與純菌株污染物處理研究觀點以及技術上都有很大的差異,未來如果可以將純菌株的概念加入到混合菌生物復育應用的過程加以探討,相信將會有新的概念及理論產生,並且可以以最簡化的程序、最節約的成本以及最少的人力達成環境污染物生物復育的效果。
Bioremediation technology has fast developed over the last 30 years in many industrialised countries. However, the rate and the extent of development has baffled by unexpectable relationships between microorganisms. A successful bioremediation scheme relies on the management of soil microbial populations capable of catabolising the contaminants. The role of soil microbiota in the biochemical conversion of organic and inorganic contaminants has been realised, priority research needs have been identified and effort has been made to understand the ecological, biochemical and genetic basis of microbial contaminant degradation, with a view to enhancing microbial capabilities and thus designing more effective bioremediation processes. Previous reports have shown that sulfate-reducing or iron-reducing bacteria could oxidize simple organic molecules using the sulfate or iron ion as an electron acceptor. This process produces hydrogen sulfide (H2¬S) and the bicarbonate ion (HCO3-). Hydrogen sulfide readily reacts with heavy metal ions (iron) to immobilize the metals as insoluble metal sulfides, while the bicarbonate ions buffer the pH to significantly higher levels (Dvorak et al. 1992). Thus, sulfate is removed as hydrogen sulfide gas and immobilized metal sulfides, metals are removed as metal sulfides, while pH is raised, improving water quality. In order to maintain bacterial metabolism, the bacteria must be given both an organic carbon source and some times substrate for attachment. Although these microorganisms can degrade organic contaminants in polluted aquifers, the process can be slow. In this thesis, we have demonstrated that sulfate-reducing bacteria can dehalogenate chlorinated aromatic compounds and iron-reducing bacteria can decolozed and detoxified dyes of different chemical structure under different incubation conditions, e.g. under different pH, temperature, electron donors or electron acceptors. In order to further characterization of these anaerobic microbial communities, physiological analysis, biochemical analysis, traditional and molecular biotechnology, for microorganism isolation and characterization are needed to fully understand the role of these microorganisms in the biodegradation of contaminants in the environments. In pentachlorophenol biodegradation study, PCR and primers specific to several dechlorinating bacteria, e.g. Desulfomonile, Desulfotomaculum, Desulfuromonas, Dehalococcoides, Desulfitobacterium, and Desulfovibrio were used to detect the presence of these dechlorinating bacteria during reductive dechlorination of pentachlorophenol (PCP) in estuarine sediment slurries. PCP dechlorination rates in anoxic sediment slurries amended with lactate plus sulfate were about eight times higher than those in anoxic sediment slurries amended with lactate plus sulfite or pyruvate, or without any amendment. Gene sequences of Desulfomonile and Desulfotomaculum were not present, while gene sequences of Desulfuromonas and Desulfovibrio were present in all tested sediment slurries. Gene sequences of Dehalococcoides and Desulfitobacterium were found in sediment slurries without any amendment. When under methanogenic and pyruvate fermentation conditions, PCP enrichment did not change the amount of these types of bacteria, however, when sulfate or sulfite was amended the amount of these types of bacteria decreased. The clones from the genes amplified from DNA extracted from PCP dechlorinating sediment slurries amended with sulfate and lactate, and primers specific to Desulfovibrio fell into four phylogenetic lineages. Among them two clones were close related to Desulfovibrio sp. TBT-1 (12.5%) and Desulfovibrio sp. PCP-1 (12.5%), respectively. In the second study, in order to study the presence or the effects of sulfate reducing bacteria (SRB) in the heat exchange cooling system of a petroleum refinery, water and sediment were sampled from 7 sites of the heat exchange cooling system to investigate the relationship between SRB population and the microbiologically influenced corrosion. Desulfotomaculum was found in sediments collected from the carbon steel condenser and the bottom tank of tower number 7, while Desulfovibrio was found in sediment of carbon steel condenser. Results of most probable number (MPN) tests also showed that the total number of microorganism ranges from 106 to 107 cells / 100 ml and the number of SRB is lower than 2 × 103 cells / 100 ml. One SRB strain was isolated from a carbon steel condenser in this study. 16S rDNA sequence analysis attested that this strain is closely related to gram negative bacterium Desulfovibrio vulgaris subsp. vulgaris str. Hildenborough. This strain can couple ferrous iron oxidation with sulfate reduction. Based on the results of SRB isolation, most probable number (MPN) analysis and the biocorrosion states of sampling sites it seems that SRB did not play a major role in biocorrosion of heat exchange cooling system. An iron-reducing bacterium, strain NTOU1, was isolated from the precipitate suspension collected from a heat exchange cooling system of a refinery plant in Taoyuan, in northern Taiwan. Cells of this strain were Gram-negative, rod-shaped, pink to orange-pigmented and motile with a single polar flagellum. No endospores are formed. This strain can grow under aerobic conditions. It was not able to ferment glucose, but was capable of anaerobic growth utilizing a variety of electron acceptors, including Fe(III), Mn(IV), iron oxide, sulfate, sulfite, thiosulfate, nitrate, nitrite, arsenate selenate, and selenite. Lactate, pyruvate, formate and H2 were used as carbon and energy sources. Physiochemical tests showed that this strain is a facultative, catalase-, oxidase-, amylase-, urease-, gelatinase- and sulfite reductase-positive, lipase-negative bacterium. The orange to pink pigment of this strain has absorption peaks at 342 and 396 nm. It grows at 10-37oC, pH 5-9 and NaCl concentration from 0 up to 5.5%. The predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8. The major fatty acid compositions of the membrane lipids were 16:0, iso-15:0, 16:1ω7c, and 17:1ω8c. The DNA G+C content of this strain was 50.3 mol%. 16S rRNA gene sequence analysis indicated that this strain has 97% and 96% similarity with Shewanella decolorationis and Shewanella putrefaciens, respectively. The gyrB gene sequence analysis also indicated that this strain has 97% and 86% similarity with S. decolorationis and S. putrefaciens, respectively. DNA-DNA hybridization showed relatedness values of 74.4% with S. decolorationis S12T. Despite a number of phenotypic and physiological differences between strain NTOU1 and S. decolorationis we propose including strain NTOU1 as a subspecies of S. decolorationis for which we propose the name Shewanella decolorationis subspecies taiwanensis. The type strain is NTOU1 (=BCRC 910321T =JCM 14211T). S. decolorationis NTOU1 which could decolorize a range of azo dyes (congo red, sudan black. acid orange, orange G, methyl orange, and methyl red) at high efficiency without a mediator. The most suitable pH values and temperatures for decolorization are pH 6.0-7.0 and 30-40oC. Decolorization rates are highest when H2, formate, or pyruvate is used as the electron donor. Addition of ferric citrate, manganese oxide, selenate, nitrate, ferric oxide, or thiosulfate increases decolorization rates of congo red by this strain. It seems that when the incubation condition is changed to aerobic condition after decolorization of congo red under anaerobic condition, benzidine, anaerobic decolorization product is further degraded under aerobic conditions by this species. S. decolorationis NTOU1 also could decolorize a range of triphenylmethane dyes without a mediator. Crystal violet could be decolorized by this species under anaerobic, but not under aerobic condition. The most suitable pH values and temperatures for decolorization of crystal violet were pH 8-9 and 30-40oC, respectively. Formate (20 mM) was the better electron donor, while nitrate (20 mM) and ferric citrate (20 mM) were the better electron acceptors for this strain to decolorize crystal violet under anaerobic conditions. By supplementing the anoxic phosphate buffered medium (pH 8) with formate (20 mM) and ferric citrate (20 mM) and cultivating it at 35 oC, this strain could decolorize these above mentioned dyes (200 mg l-1) within 2-11 h under anaerobic conditions, with the initial color removal rates being: malachite green > crystal violet > methyl violet B > basic fuchsin > bromophenol blue. By supplementing the medium with formate and ferric citrate and cultivating it under optimum pH and temperature, this strain could remove crystal violet, at a concentration of 1500 mg l-1, at the rate of 298 mg l-1 h-1 (during decolorization the OD600 of the cell culture increased from ~0.6 to ~1.2). GC/MS analysis of the degradation products of crystal violet detected the presence of N,N’-bis(dimethylamino) benzophenone (Michler’s Ketone), [N,N-dimethylaminophenyl] [N-methylaminophenyl] benzophenone, N,N-dimethylaminobenzaldehyde, N,N-dimethylaminophenol, and 4-methylaminophenol. These results suggest that crystal violet was biotransformed into N,N-dimethylaminophenol and Michler’s Ketone prior to further degradation of these intermediates. This thesis proposes probable pathways for the degradation of crystal violet by this Shewanella sp. Cytotoxicity and antimicrobial tests showed that the process of decolorization also detoxify crystal violet. GC/MS analysis of the intermediate compounds during decolorization and degradation of malachite green and methyl violet B detected leucomalachite green, N,N,N’-trimethyl-4,4’-benzylidenedianiline, [N,N-dimethylaminophenyl] [phenyl] benzophenone, and N,N-dimethylaminophenol, and leucomethyl violet B, N,N’-bis [dimethylamino] benzophenone (Michler’s ketone), [N,N-dimethylaminophenyl] [N’-methylaminophenyl] benzophenone, N,N-dimethylaminobenzaldehyde, N,N-dimethylaminophenol, N-methylaminobenzaldehyde, and N-methylaminophenol, repectively. Probable pathways for the decolorization and degradation of malachite green and methyl violet B by this strain were proposed based on the time course of degradation of parent compound and sequential biotransformation of intermediate compounds. Cytotoxicity antimicrobial tests showed that the toxicity of malachite green or methyl violet was slightly decreased just after decolorization, however, along with further incubation, the toxicity increased again. Shewanella sp. NTOU1 was able to decolorize a range of anthraquinone dyes [Reactive Blue 4 (RB4), Reactive Blue 19 (RB19), Mordant Red 11 (MR11), Disperse Red 15 (DR15), and Disperse Blue 3 (DB3)] under anaerobic conditions. By supplementing the medium with formate and ferric citrate as the electron donor and acceptor, respectively and cultivating it under the optimum pH (8.0-9.0) and temperature (45 oC), this strain could decolorize these dyes (1000 mg l-1) at the initial color removal rates of 15-126 mg l-1 h-1 and the rates among them were RB19 > RB4 > DB3 > DR15 > MR11. The extent of color removal of these dyes was more than 39% at a dye concentration of 1000 mg l-1. 1-aminoanthraquinone, 2,3-dihydro-9,10-dihydroxy-1,4-anthracenedione, and leuco-1,4-diaminoanthraquinone were detected with GC/MS after color removal from both RB4 and RB19, while 1-hydroxy-9,10-anthracenedione was detected from MR11, 1,4-dihydroxy-9,10-anthracenedione and 2,3-dihydro-9,10-dihydroxy-1,4- anthracenedione were detected from DR15, and leuco-1,4-diaminoanthraquinone was detected from DB3. Based on the decolorization products, probable pathways for the decolorization of these dyes by this strain were proposed.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0D90340001
http://ntour.ntou.edu.tw/ir/handle/987654321/17422
Appears in Collections:[海洋生物研究所] 博碩士論文

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