利用饋料批式靜態培養Komagataeibacter xylinus優化細菌纖維素產量

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Title:	利用饋料批式靜態培養Komagataeibacter xylinus優化細菌纖維素產量 Optimizing the production of bacterial cellulose by statically cultured Komagataeibacter xylinus using fed-batch fermentations
Authors:	劉柏廷 LIU, PO-TING
Contributors:	化學工程與材料工程學系
Keywords:	細菌纖維素靜態培養饋料批式發酵回收流發酵過氧化鈣發泡煉石 bacterial cellulose static culture fed-batch fermentation recycle stream fermentation calcium peroxide lightweight expanded clay aggregate
Date:	2024
Issue Date:	2024-11-14 10:54:17 (UTC+8)
Abstract:	細菌纖維素(Bacterial cellulose, BC) 是細菌一次代謝所分泌之胞外纖維，BC因具備特殊的立體網狀奈米結構，在固有的特性上存在不少競爭優勢，如高保水性、高生物相容性、生物可分解性、高機械強度等。目前已有許多BC的應用實例，如生醫產業上的生物纖維面膜、人工敷材、人工血管。而多數BC相關產品需仰賴靜態培養之薄膜型態，但靜態培養期間細菌生長條件難以控制，使得提升BC產量為受關注的課題。本實驗透過市售玻璃茶壺作為靜態培養系統，主要以饋料批式培養、回收流培養、添加過氧化鈣及添加發泡煉石(Lightweight expanded clay aggregate, LECA)四大主軸進行BC產量的優化。實驗結果顯示，側管法饋料批式培養能有效解決初始糖濃度高不利於BC合成的問題，使原先初始葡萄糖濃度20g/L與初始葡萄糖濃度30g/L差1.5倍，但實際BC產量不符合碳源比例的議題，透過側管法饋料批式及調控培養基pH值操作培養下，將BC產量達趨近理想值的1.41倍。而隨著細菌的生長與BC持續合成而使膜愈來愈厚時，便會產生明顯的質傳阻力，導致膜下方的培養基養分會愈來愈難穿越厚膜達到膜表層供菌體使用，透過回收流培養使15天培養週期中，以克服養分質傳的方式無本提升BC產量18%。實驗上也針對靜態培養之培養基溶氧耗罄的問題，透過茶壺側管於第4天添加過氧化鈣給予菌體溶氧，經調控培養基pH值操作培養下BC產量可提升至18%。最後以LECA孔隙攜帶氣體的特性及釋鹼成分，置於培養基當中，提供菌體有較佳的生長環境，使BC產量提升約20%，解決溶氧低下及培養基酸性代謝物積累不利BC合成的問題。在BC材料分析方面，側管法饋料批式培養並未改變BC官能基，結晶度約86%，SEM成像BC密集而相連。而回收流培養也未改變BC官能基，結晶度約88%，透過回收流操作使BC的SEM成像網絡結構不明顯。添加過氧化鈣培養也未改變BC官能基，結晶度約87%，SEM成像BC明顯變粗且密度高。添加LECA培養也未發現新的官能基，但官能基強度明顯較弱，結晶度約87%，SEM成像BC具包覆感而不明顯。本實驗藉由茶壺系統的特性，提供不同的靜態培養策略，優化細菌生長條件並提升BC產量，有利於生產上的應用。 Bacterial cellulose (BC) is an extracellular fiber secreted by bacteria through primary metabolism. BC has a special three-dimensional network nanostructure and has many competitive advantages in inherent characteristics, such as high water retention and high biophase. Capacitance, biodegradability, high mechanical strength, etc. There are currently many application examples of BC, such as biofiber masks, artificial dressings, and artificial blood vessels in the biomedical industry. Most BC-related products rely on the film type of static culture. However, bacterial growth conditions are difficult to control during static culture, making increasing BC production a topic of concern. This experiment uses a commercially available glass teapot as a static culture system, and mainly uses four main axes of feeding batch culture, recycling flow culture, adding calcium peroxide, and adding lightweight expanded clay aggregate (LECA) to measure BC production. optimization. Experimental results show that the side-tube feeding batch culture can effectively solve the problem of high initial sugar concentration that is not conducive to BC synthesis. The original initial glucose concentration of 20g/L is 1.5 times different from the initial glucose concentration of 30g/L, but the actual BC yield is not In line with the issue of carbon source ratio, through the side-tube method of feeding batches and adjusting the pH value of the culture medium, the BC yield is 1.41 times closer to the ideal value. As bacteria grow and BC continues to be synthesized, and the membrane becomes thicker and thicker, significant mass transfer resistance will occur, making it increasingly difficult for the culture medium nutrients under the membrane to pass through the thick membrane and reach the surface of the membrane for use by the bacteria. Through recycle stream culture, BC yield can be increased by 18% cost-effectively in a 15 days culture cycle by overcoming nutrient mass transfer. Experiments also aimed at the problem of running out of dissolved oxygen in the static culture medium. Calcium peroxide was added through the side tube of the teapot on the 4th day to provide dissolved oxygen to the bacteria. By adjusting the pH value of the medium, BC production could be increased to 18 %. Finally, the gas-carrying characteristics of LECA pores and the alkali-releasing components are placed in the culture medium to provide a better growth environment for the bacteria, increase BC production by about 20%, and solve the problem of low dissolved oxygen and the accumulation of acidic metabolites in the culture medium that are detrimental to BC synthesis problem. In terms of BC material analysis, the side-tube feeding batch culture did not change the BC functional groups, the crystallinity was about 86%, and the BCs were dense and connected in SEM imaging. The recycling flow culture did not change the functional groups of BC, and the crystallinity was about 88%. The SEM imaging network structure of BC was not obvious through the recycle stream operation. Adding calcium peroxide to culture did not change the functional groups of BC. The crystallinity was about 87%. BC was obviously thicker and denser in SEM imaging. No new functional groups were found after adding LECA to the culture, but the strength of the functional groups was obviously weak, the crystallinity was about 87%, and the SEM image of BC had a coating feeling but was not obvious. This experiment uses the characteristics of the teapot system to provide different static culture strategies to optimize bacterial growth conditions and increase BC production, which is beneficial to production applications.
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