以 Yarrowia lipolytica 從甘油進行丁四醇轉換生產之策略

Abstract

本研究於不同生物反應器中進行以 Yarrowia lipolytica Po1g 將甘油轉換成丁 四醇策略之研究。過去之研究中，並沒有比較過不同生物反應器策略，而根據過 去文獻所使用之醱酵槽策略， 本研究提出培養基置換(growth medium replacement, GMR) 與鹽類外添加(growth medium salt adding, GMS) 兩種搖瓶培 養策略，先將Y. lipolytica 接種於適合菌體累積之growth medium (GM) 培養基中 進行生菌數累積，當菌體累積至定常期時，再將菌體置入適合丁四醇轉換之 Erythritol Synthesis Medium (ESM) 培養基，或是以鹽類添加之方式將環境滲透壓 提高，以進行丁四醇之轉換。上述兩種培養策略分別能在搖瓶培養96 小時後累 積8.19、7.61 g/L 之丁四醇。然而，當直接以ESM 培養基進行Y. lipolytica 培養 時，雖然生菌數較上述兩種策略低，但所能產生之丁四醇含量到達14.56 g/L，仍 較上述兩種策略為高。本研究也以海藻酸鈣包埋法將Y. lipolytica 固定化後進行 丁四醇連續轉換。當以固定化膠粒於搖瓶中進行丁四醇轉換時，雖然能夠成功轉 換出丁四醇，但固定化之膠粒結構可能會在振盪培養的過程中被損壞，使固定化 膠粒再次利用之效能大幅降低。而若將固定化膠粒填充於管柱型生物反應器中之 醱酵反應，推測因為溶氧不足使固定化膠粒無法進行丁四醇轉換。本研究最後探 討接種量對醱酵槽批次饋料培養Y. lipolytica 進行丁四醇轉換時之影響。結果發 現，當接種量從0.1 % 提升至10 % 時，可以提高生菌數之累積速度，也提升Y. lipolytica 的丁四醇含量，使菌體在第324 小時之丁四醇累積量從58.61 g/L 提升 33.61% 至78.31 g/L，而丁四醇之產率也從0.18 g/L·h 提升至0.24 g/L·h。而此一 提升有可能是起因於種菌培養的GM 培養基成份所造成。而10 % 接種量之批次 饋料培養能在收槽時(第552 小時) 將丁四醇含量累積至119.40 g/L。因此，醱 酵槽批次饋料培養仍是比較好的丁四醇轉換策略。

In this study, we study the ability of Yarrowia lipolytica Po1g for erythritol conversion from glycerol by different kinds of bioreactors and strategies. There was no studies about different bioreactor strategies before, and based on previous fermentation research, two flask culture strategies, growth medium replacement (GMR) and growth medium salt adding (GMS), are used in this study. At first, Y. lipolytica is inoculated in growth medium (GM), which is beneficial for yeast growth, to accumulate yeast cells. When yeast cells enter stationary phase, transfer Y. lipolytica cells into Erythritol Synthesis Medium (ESM), which is suitable for Y. lipolytica to produce erythritol, or raise the osmotic pressure by adding extra salt to make Y. lipolytica start the conversion of erythritol. The production of erythriol reached 8.19, 7.61 g/L separately after 96 hours culture by the two strategies above. However, if culture Y. lipolytica in ESM medium directly, the production of erythritol reached 14.56 g/L was higher than the production of the two strategies. Yeast immobilization entrapped in calcium alginate for the conduction of continuous fermentation is also studied. Though it’s executable for the Y. lipolytica immobilized gels to produce erythritol, the production rate decreased drastically after reusing. It may be because of the destruction of gels structure during the shaking culture. Also, the conversion of erythritol by filling immobilized gels into column bioreactor cannot be detected. Without efficient oxygen for erythritol conversion may be the main reason. The effect of inoculum amount to erythritol production in Y. lipolytica fed-batch culture was discussed in this study. The result showed that when the inoculum amount was elevated from 0.1% to 10%, it would not only promote the growth rate of Y. lipolytica but also improve the erythritol production, which the concentration of erythritol increased 33.61% from 58.61 g/L to 78.31 g/L andthe erythritol production rate also increased from 0.18 g/L·h to 0.24 g/L·h. This increment may be due to the element from inoculum medium, GM medium. And the erythritol amount reached 119.40 g/L after 552 hours cultivation in 10 % inoculum fedbatch culture. Thus, fermentation bioreactor fed-batch culture was still the better strategies for erythriol conversion.