Effect of different layered double hydroxides addition on surfactin production by Bacillus subtilis CWS1 in the submerged culture

近年來，生物界面活性劑surfactin（從自然中生產之胜肽），被廣泛地運用在生物醫學上。雖然生物界面活性劑在生物醫學與植物病學上極具潛力，但量產及純化條件的資訊仍是有限的。先前本實驗室的結果指出以枯草桿菌Bacillus subtilis CWS1液態醱酵生產表面素surfactin，添加層狀雙氫氧化物Mg-Al-nitrate LDHs可以有效增加表面素之產量。 本研究將分為兩部分作探討，第一部分針對其吸附機制作探討，由XRD分析 LDHs層間距由吸附前的7.8 Å撐開至16.1 Å，藉此證實層狀雙氫氧化物會將表面素吸附其中，同時以HPLC分析LDHs之內含物，發現具有表面素之存在，並藉由SEM與EDX成分分析可觀察層狀雙氫氧化物吸附情形。 第二部分將針對不同比例之Mg-Al-nitrate LDHs以及Mg-Fe-nitrate LDHs添加對於枯草桿菌Bacillus subtilis CWS1發酵產表面素產量之探討，結果表示，添加6 g/L濃度的Mg-Al-nitrate LDHs主培養五天可得到表面素最大產量約3789 mg/L；添加6 g/L濃度的Mg-Fe-nitrate LHDs主培養第一天就產出4280 mg/L的表面素，四天即可產5130 mg/L的表面素，顯示用Mg-Fe-nitrate LDHs對於CWS1產表面素之效益更加突出，並針對菌量與表面素產量作與時間的關係圖，顯示菌體對於此添加物有很好的適應性，但是其產表面素能力依舊維持一動態平衡；經一系列實驗探討，顯示添加Mg-Fe-nitrate LDHs有助於表面素生產之改善，並具有工業應用之潛能。Surfactin, one of the biological surfactants was found to possess great potential in biomedical and plant disease application. However, information about the production and purification condition is still limited. In the previous study, the addition of Mg-Al-nitrate LDHs in the liquid culture of Bacillus subtilis CWS1 could significantly increase the surfactin production This study includes two part. In the first part, XRD (X-ray diffractometer) was used to measure the basal spacing of LDHs. The surfactin intercalation involves an ionic exchange reaction of LDHs at 25℃ and under N2 atmosphere in the aqueous solution could yield a series of biomolecules-LDHs with basal spacing ranging from 7.8 Å of 16.1 Å, revealed by XRD analyses. The wide interlayer spacing might be attributed to the self-alignment of the biomolecules in the layer confinement, as analyzed the components by HPLC. In addition, SEM (scanning electron microscope) and EDX (Energy-dispersive X-ray spectrometry) was used to verify the adsorption of surfactin. The process of embedding biomolecules into the clay gallery provides a new method for synthesizing biomaterial/LDHs hybrids potentially useful in agriculture or biomedical design. In the second part, different LDHs, Mg-Al-nitrate LDHs and Mg-Fe-nitrate LDHs, were used as additive to the cultivation of Bacillus subtilis CWS1. In our study, 6 g/L of Mg-Al-nitrate LDHs addition gave the maximum surfactin production of 3789 mg/L after five days culture. However, 4280 mg/L of surfactin was produced in one day culture when 6 g/L of Mg-Fe-nitrate LHDs was added to the cultivation. The maximum production of 5130 mg/L was obtained in four days cultivation. These results indicated that addition of Mg-Fe-nitrate LHDs in Bacillus subtilis CWS1 culture could effectively enhance the production of surfactin. As seen from the time course of the cultuvation, we could found out Mg-Fe-nitrate LHDs addition was found harmless to Bacillus subtilis, the production of surfactin was kept in a dynamic equilibrium. This study provided an approach for improving surfactin production from Bacillus subtilis CWS1 via the addition of Mg-Fe-nitrate LDHs as a stimulator.總目錄 摘要 I Abstract II 總目錄 IV 表目錄 VI 圖目錄 VII 第一章、緒論 1 1.1 前言 1 1.2研究動機 1 第二章、文獻回顧 2 2.1 枯草桿菌(Bacillus subtilis) 2 2.2 表面素(surfactin) 4 2.2.1生物界面活性劑 4 2.2.2 抗菌脂質胜肽(lipopeptide) 10 2.2.3表面素之結構性質 10 2.2.4 表面素之研究與應用 11 2.3 微生物製劑 13 2.4 層狀雙氫氧化物 16 2.4.1雙層結構 16 2.4.2備製方法與原理 18 2.4.3層狀奈米複合物 20 2.5 吸附 22 2.5.1物理性吸附 22 2.5.2化學吸附 22 2.5.3交換吸附 22 第三章、材料與方法 24 3.1 實驗藥品 24 3.2 儀器設備 26 3.3 實驗菌種 28 3.4枯草桿菌(Bacillus subtilis CWS1)生產surfactin之方法 28 3.4.1 平板活化 28 3.4.2 前培養 28 3.4.3 主培養 28 3.5 層狀雙氫氧化物製備(共沉澱法) 30 3.5.1 Mg-Al-nitrate-LDHs製備 30 3.5.2 Mg-Fe(III)-nitrate-LDHs製備 31 3.5.3 固含量測試 32 3.6 實驗分析方法 33 3.6.1 實驗分析 33 3.6.2 LDH之吸附實驗方法 35 3.6.3 LDH之酸崩解方法 35 3.6.4 LDH之XRD(X-Ray Diffraction)層間距分析 36 3.6.5 菌體濃度測試 37 3.6.6 表面素之HPLC分析 37 3.6.7 能量色散X-射線光譜分析 39 第四章、結果與討論 40 4.1 不同重量比例之Mg-Al LDHs 吸附surfactin之比較 40 4.1.1表面素與LDHs不同重量比例之插層探討 40 4.1.2 水氣對於表面素插層LDHs之探討 43 4.1.3表面素組成對於其插層LDHs之探討 43 4.2 LDHs對於Bacillus subtilis CWS1生產表面素之影響 45 4.2.1添加物對於Bacillus subtilis CWS1生產表面素探討 45 4.2.2添加Mg-Al-nitrate LDHs之濃度對於CWS1生產表面素探討 47 4.2.3添加不同離子比例Mg-Al-nitrate LDHs對於CWS1生長探討 50 4.2.4添加Mg-Fe-nitrate LDHs之濃度對於CWS1生產表面素探討 53 4.2.5添加不同離子比例Mg-Fe-nitrate LDHs對於CWS1生長探討 56 4.2.6添加不同LDHs對於CWS1生長型態影響 59 第五章、結論與未來展望 63 5.1結論 63 5.2未來展望 6