以Gluconacetobacter xylinus生產細菌纖維素之研究

Abstract

細菌纖維素是由特殊的菌株所分泌的一種胞外不可溶性多醣類，因為其超微細網狀結構及其它特性，在工業的應用上具有相當大的前景，如造紙紡織、食品工業以及生醫材料等。本研究主要探討不同的環境因子以及改變培養基配方對於Gluconacetobacter xylinus生成纖維素產量的影響。 因為纖維素的型態會隨著培養方式以及培養基的配方而有所差異，且若未先有效的去除內部的雜質，對於纖維素產量的估算會造成很大的影響。本研究建議應先經由剪碎的程序，再經過0.5N NaOH熱鹼處理30分鐘後，藉此將包含在纖維素團塊內部的不純物質移除而得到較為合理且穩定的纖維素乾重。 本研究以BSH Medium作為培養基(初始pH值調整至5.5)，在26℃下以迴旋式(100rpm)進行7天培養所得的纖維素產量作為對照組(2.5g/L)，分別探討不同的環境因子對於纖維素產量的影響。由實驗結果可以發現，當初始pH值設定在6.0~7.0之間進行培養，則會有較高的纖維素產量(3.5g/L)。而改用往復式培養(100rpm)或是靜置培養的組別所得的纖維素產量較低，所生成的纖維素型態也大不相同，因此之後的實驗條件皆選擇迴旋式(100rpm)進行培養。 另一方面，藉由改變培養基中碳、氮源的種類以及濃度，可以有效的提高纖維素的產量。在碳源的選擇上，分別利用2%的蔗糖、葡萄糖、果糖、甘露醇作為單一碳源時皆可得到1.5g/L以上的產量，其中以蔗糖作為碳源時有最高的纖維素產量(3.53g/L)。此外，提高葡萄糖的濃度，纖維素的產量也會隨之提升，當葡萄糖的濃為80g/L時，纖維素產量可以達到5.0g/L。而在氮源的選擇上，測試了酵母萃取物、蛋白腖、玉米浸漬液作為調控的主要氮源。其中添加60g/L的玉米浸漬液的組別有最高的纖維素產量(13.56g/L)。同時添加其他的生長因子，如乙醇、有機酸等皆有助於纖維素產量的提升，其中額外添加5g/L的延胡索酸於培養基當中，亦可以有效提升纖維素的產量(7.0g/L)。

Bacterial cellulose (BC) is a kind of insoluble extracellular polysaccharide produced by Gluconacetobacter strains. Because of the unique properties, mainly the ultrafine reticulated structure, BC has found various applications in paper, textile, and food industries, and as a biomaterial in cosmetics and medicine. In this study, we investigated different cultural methods/conditions and medium composition to understand their influence on the BC production with Gluconacetobacter xylinus. We found that the morphology of BC depends on the cultural methods and composition, and significant amount of medium might be entrapped inside the BC. If we do not remove that out of BC, the BC yield might be overestimated. Therefore, we suggest BC be cut into pieces to release the medium before treating with 0.5N NaOH at 90℃ for 30mins to release the rest of impurities. When using BSH Medium (initial pH: 5.5) at 26°C under orbital shaking (100rpm), BC yield was 2.5g/L after 7 days cultivation. Therefore, we set it as a control group to discuss the effect of different environmental factors on the BC production. We tested BC yield for the initial pH ranging from 3.5 to 7.0, and found a maximum between pH 6.0 and 7.0 (3.5g/L). Besides, BC yield under orbital shaking (100rpm) is the highest compared with static cultivation and reciprocal shaking (100rpm). To investigate the effect of carbon sources on the production of BC, various carbon sources were investigated at 2%(w/v). Good BC production was obtained when sucrose, glucose, fructose, or mannitol was used as the sole carbon source, and the highest yield was obtained with the sucrose (3.53g/L). Furthermore, BC yield would become higher with increasing glucose concentration, and the highest yield occurred at 8.0% (5.0g/L). On the other hand, various nitrogen sources such as yeast extract, peptone, and corn steep liquor (CSL) were tested to assess their effects on BC production, and the highest cellulose yield was obtained for the case of 6.0% corn steep liquor (13.56 g/L), which was about four times higher than in BSH Medium. Besides, other growth factors, such as ethanol, organic acid, and gluconic acid were also found to stimulate the BC production. For example, with 0.5% fumaric acid addition, the BC yield could increase from 2.5g/L to 7.0g/L.