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標題: 好氣式堆肥嗜高溫菌之調查與分解纖維之新菌株研究
Investigation of Thermophilic Bacteria in Aerobic Composts and Characterization of a Novel Cellulose-degrading Isolate
作者: 王昭閔
Wang, Chao-Min
關鍵字: aerobic compost;好氣式堆肥;thermophilic bacteria;cellulose degrading;bacillus;嗜高溫菌;纖維分解;芽胞桿菌
出版社: 獸醫微生物學研究所
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本研究分析16S rDNA序列及Biolog生化資料,探討在禽糞及牛糞堆肥之嗜高溫菌的菌相及其利用之物質。此外,在研究過程中發現一個會分解纖維素(cellulose)的嗜高溫Paenibacillus新菌株。根據16S rDNA序列分析建構親緣演化關係,本研究自好氣式堆肥分離之34株嗜高溫菌(培養於60°C)分別鑑定為Bacillus licheniformis、B. atrophaeus、Geobacillus stearothermophilus、G. thermodenitrificans、Brevibacillus thermoruber、Ureibacillus terrenus、U. thermosphaericus及Paenibacillus cookii。其中,B. atrophaeus、Br. thermoruber及P. cookii為首次自高溫堆肥中分離之菌種。而根據Biolog GP系統所提供之菌種生化反應資料,以主成分分析法(principal component analysis, PCA)分析後可將菌種區分為8個不同之PCA族群。自禽糞堆肥中分離之嗜高溫菌共21株,分屬於7種菌種及8個PCA族群,較牛糞堆肥來源菌種(分離13株,5種菌種及4個PCA族群)複雜。其中B110菌株(自禽糞堆肥分離)與U. terrenus之16S rDNA序列相似度達97.6%,並且會利用許多種物質,是一株具有工業利用價值之菌株。此外,在經過篩選後,我們發現一株具有纖維素分解能力之嗜高溫Paenibacillus新菌株B39。B39與P. cookii在16S rRNA基因序列極為相似,然而在許多生理特徵、生化反應、菌種脂肪酸構成及抗藥性方面都不盡相同,因此應為一株新的Paenibacillus菌株。經由離子交換層析法純化得到B39菌株分泌之纖維分解酵素(cellulase),此酵素分子量約為148 kDa,在60°C、pH6.5條件下,具有同時分解水溶性羧甲基纖維素(carboxymethylcellulose, CMC)與微結晶狀纖維素(microcrystalline cellulose)(Avicel)之最大活性。添加1 mM Ca2+離子可同時增加其羧甲基纖維素酶(CMCase)與微結晶纖維素酶(Avicelase)之能力。總而言之,於本研究我們詳細調查一株新的嗜高溫Paenibacillus菌株B39,此菌株會分泌一種兼具CMCase與Avicelase活性的高分子量cellulase。

This study investigated species diversity and substrate utilization patterns of culturable thermophilic bacterial communities in hot aerobic poultry and cattle manure composts by coupling 16S rDNA analysis with Biolog data. In addition, a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39 was identified and characterized. Based on the phylogenetic relationships of 16S rDNA sequences, 34 thermophilic (grew at 60C) bacteria isolated during aerobic composting of poultry manure and cattle manure were classified into Bacillus licheniformis, B. atrophaeus, Geobacillus stearothermophilus, G. thermodenitrificans, Brevibacillus thermoruber, Ureibacillus terrenus, U. thermosphaericus, and Paenibacillus cookii. In this study, B. atrophaeus, Br. thermoruber and P. cookii were reported for the first time in hot compost. Physiological profiles of these bacteria, obtained from the Biolog Gram-positive (GP) microplate system, were subjected to principal component analysis (PCA). All isolates were categorized into eight different PCA groups based on their substrate utilization patterns. The bacterial community from poultry manure compost comprised more divergent species (21 isolates, 7 species) and utilized more diverse substrates (8 PCA groups) than that from cattle manure compost (13 isolates, 5 species, and 4 PCA groups). Many thermophilic bacteria isolated in this study could use a variety of carboxylic acids. Isolate B110 (from poultry manure compost), which is 97.6% similar to U. terrenus in its 16S rDNA sequence, possesses particularly high activity in utilizing a broad spectrum of substrates. This isolate may have potential applications in industry. On the other hand, after screening the 34 thermophilic bacteria mentioned above, we detected cellulolytic activity in a novel thermophilic Paenibacillus sp. strain B39. Strain B39 was closely related to P. cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel strain of Paenibacillus sp. according to its physiological characteristics, biochemical reactions, the profiles of fatty acid compositions and the resistance spectrum to antibiotics. A cellulase secreted from strain B39 was purified by ion-exchange chromatography. The molecular weight of B39 cellulase was determined as 148 kDa. B39 cellulase exhibited the ability to hydrolyze both carboxymethylcellulose (CMC) and microcrystalline cellulose (Avicel), with a maximum CMCase activity at 60C and pH 6.5. Addition of 1 mM Ca2+ markedly enhanced both CMCase and Avicelase activities of the enzyme. In conclusion, we have characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high molecular weight cellulase with both CMCase and Avicelase activities.
其他識別: U0005-3005200817204600
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