Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/25631
標題: 根瘤菌胞外多醣產量優化及其功能與特性之研究
Optimization for production and functional characterization of exopolysaccharides from rhizobia
作者: 黃國鑫
Huang, Kuo-Hsin
關鍵字: 根瘤菌;rhizobial;胞外多醣;乳化指數;生物分析;exopolysaccharide;emulsification index;bioassay
出版社: 土壤環境科學系所
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摘要: 
利用根瘤菌來進行土壤及水域中有機污染物的降解,是一種新的嘗試與作法。本研究自實驗室菌種庫挑選經16S rDNA鑑定之40株根瘤菌,進行胞外多醣及胞外分泌物之柴油乳化指數E24(%)測試,得到具高柴油乳化能力及較佳穩定性之三株根瘤細菌Rhizobiummiluonense CC-B-L1、Burkholderiaseminalis CC-IDD2w及Ensiferadhaerens CC-GSB4,以YEM培養基培養測定其胞外多醣(EPS)之含量,分別為212 mg L-1、203 mg L-1及198 mg L-1,柴油E24乳化指數分別為65.5%、63.9%及59.8%。調整YEM培養基成分對於根瘤菌之胞外多醣產量,顯示添加1.5%甘露醇和0.1%天門冬素對於CC-B-L1及CC-IDD2w;添加2.0%葡萄糖和0.2%天門冬素對於CC-GSB4,其產量分別增加30.0、33.1%及39.5%,對於柴油E24分別提升23%及27.1%及27.3%。
其次,菌株胞外分泌物與胞外多醣功能試驗,菌株CC-IDD2w具有溶磷活性;三株菌的胞外多醣,在不同油品之乳化效能測試,除了汽油乳化能力較差,食用大豆油、機油和柴油皆有良好乳化能力。胞外多醣酪胺酸酶抑制試驗,以菌株CC-B-L1之胞外多醣,具有較高的酪胺酸酶活性抑制率,其次是CC-IDD2w、而CC-GSB4之胞外多醣沒有抑制作用,本研究首次證實根瘤菌之胞外多醣具有美白效果。種子發芽生物分析的結果顯示,此三株根瘤菌菌株培養,對於萵苣種子胚莖均有促進效果;對於胚根生長則有抑制作用。胞外多醣元素分析顯示CC-B-L1、CC-IDD2w及CC-GSB4株菌胞外多醣之構成元素,為O/C比值較高,且具有較高的脂肪族碳,與成份中有較高的極性與非極性比值,可作為推論有較佳油品乳化效果原因。胞外多醣官能基分析在於927 cm-1和850 cm-1(β-glycosidic)、1079-1026cm-1 (pyranoside與furanoside醣苷鍵)有吸收峰,確實為胞外多醣。胞外多醣以1HNMR光譜測得3.66-3.83 ppm為非異構氫;13C-NMR光譜圖有羥基碳(63.3 ppm)及環狀碳70.8 ppm);比對圖譜化學位移H4/C4有接近β-glucuronicacid (β-GlcA)部份殘醣結構。

The use of rhizobia for degradation of organic pollutants in soil and water is a new attempt and practice. In this study, index E24 (%) and stability of diesel emulsification by extracellular secretions from 40 strains, which were identified as rhizobia based on 16S rDNA sequence, were evaluated and three rhizobial strains Rhizobium miluonense CC-B-L1, Burkholderia seminalis CC-IDD2w and Ensifer adhaerens CC-GSB4 having higher diesel emulsification capacity and stability were grown in YEM medium and exopolysaccharide (EPS) production and its diesel E24 (%) were determined as 212 mg L-1, 203 mg L-1 and 198 mg L-1, and 65.5%, 63.9% and 59.8%, respectively.
Better media composition test (YEM+1.5% mannitol+0.1% asparagine for strains CC-B-L1 and CC-IDD2w, YEM+2.0%glucose+0.2% asparagine for strain CC-GSB4) showing increasing EPS production by 30.0%, 33.1% and 39.5% and diesel E24 (%) by 23% and 27.1% and 27.3%. Extracellular secretion and EPS function tests revealed that only strain CC-IDD2w have phosphate solubilization ability; emulsification by EPS from these three strains on different oil was tested and the results showed poor emulsifying ability on gasoline, and good emulsifying ability on soybean oil, pumping oil and diesel. In whitening function tests, high inhibitory rate on tyrosinase activity was exerted by CC-B-L1 origin EPS, followed by EPS from CC-IDD2w, but no inhibitory effect by CC-GSB4 origin. This is the first evidence that rhizobial EPS having whitening effect. Seed germination bioassay using lettuce showed that the three rhizobial strains promote epicotyl growth but inhibit radicle growth. Elemental analysis of EPS indicated that EPS from strains CC-B-L1, CC-IDD2w and CC-GSB4 all have higher O/C ratio, aliphatic carbon, and polar/nonpolar ratio and these characteristics can be indexes of better oil emulsification. The functional group analysis showed a typical absorption peaks of EPS: 927 cm- 1 and 850 cm-1 (β-glycosidic), 1079 -1026 cm-1 (pyranoside and furanoside glycosidic bond). 1H NMR and 13C-NMR spectra of EPS recorded 3.66-3.83 ppm (non-heterogeneous hydrogen), and the chemical shift of 63.3 ppm (hydroxyl carbon) and 70.8 ppm (carbon ring). Chemical shift in H4/C4 indicated that the structure of EPS is close to beta-glucuronic acid (β-GlcA) residue.
URI: http://hdl.handle.net/11455/25631
其他識別: U0005-2707201220554900
Appears in Collections:土壤環境科學系

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