| 摘要: | 伴隨著工業化與科技技術的進步下，促使農業糧食生產效率更勝以往，有效滿足人類對於環境資源的需求，卻也導致大量的固體有機廢棄物產生。以台灣農業為例每年約有二十萬公噸的魚類廢棄物和十三萬公噸的蔗渣等副產物產生，過往其回收再利用，如: 青貯、堆肥和做為動物飼料中的高蛋白粉等方式，所增加的經濟價值十分有限，無法賦予更高的價值，此外，這些農業廢棄物置於環境中易因細菌發酵而產生有毒硫化氫氣體或脂肪酸經氧化反應而所散發出的油耗味。為了解決此農業廢棄物後續處理問題並達到升值再利用化，因此本篇以魚廢和蔗渣作為模式固體廢棄物，以微生物催化的方式，用以製造高價值的酮異己酸 (α-Ketoisocaproic acid, KIC)。在實驗設計中，藉由優化Bacillus subtilis分泌胞外酵素subtilisin將魚廢蛋白水解為胺基酸，可為B. subtilis提供氮源外，其中所含亮胺酸 (Leucine)佔魚廢總胺基酸的10.1%，為KIC的前驅物，本研究藉由剔除KIC後續代謝基因bkdAB累積 KIC，並透過異源表達方式誘導 L-氨基酸脫氨酶（LAAD）表達以增加KIC之產量。此外，本研究所使用的木糖來源為酸解蔗渣，相較於過往文獻，此方法能從蔗渣中獲取高濃度的木糖 (26 g/L)，並運用於誘導蛋白表達。實驗結果顯示 B. subtilis在pH 8.0、40℃下具有最佳蛋白降解速率及轉化KIC速率，且B. subtilis突變株∆bkdAB+LAAD具有最高KIC的產量，相較於B. subtilis突變株∆bkdAB多約1.75倍(3.37 mM KIC)。此外，在代謝過程中，B. subtilis突變株∆bkdAB+LAAD不僅具較高的氨基酸利用率，相較於野生株B. subtilis亦減少近半的CO2產量 (43.3%)。因此，本文成功利用微生物轉化固體有機廢棄物為無毒增值產物，不僅符合聯合國提出的Sustainable Development Goals (SDGs)中的第12項¬(有責任之消費與生產)，更與政府提出的5+2產業創新計劃中的循環經濟、低排碳相互呼應。;With the advances of industrial technology, the efficiency of agricultural production has been greatly improved. While satisfying human’s needs, the agriculture also comes along with large amounts of solid organic waste. For example, Taiwan′s agriculture produces 200,000 tons of fish waste and 130,000 tons of bagasse annually, respectively. In the past, the economic value added by organic waste recycling is low (such as compost), reducing the public’s will for organic waste recycling. Therefore, this study aims to valorize fish waste and bagasse to produce highly value-added products, and thereby enhancing the public’s will for organic waste recycling. α-Ketoisocaproic acid (KIC) is a naturally occurring metabolite of leucine that is capable of boosting animal muscle growth. In this study, we managed to develop a Bacillus-based fermentation system to produce KIC using fish waste and bagasse as the substrates. We selected Bacillus subtilis as the model organism for the KIC bioproduction because B. subtilis readily secrets extracellular protease subtilisin to the environments, which can hydrolyze fish waste protein into free amino acids. The leucine released would be subsequently converted into KIC by B. subtilis. We observed the optimal protein degradation rate in the wild-type B. subtilis cultures at pH 8.0 and at 40℃, and thus performing all the KIC bioproduction trials under such conditions. Later, we found that deletion of the bkdAB, which is responsible for KIC degradation, led to the KIC accumulation. In addition, heterologous expression of L-amino acid deaminase (LAAD) in B. subtilis increased the KIC yield up to 26 g/kg of fish waste. The results showed that the B. subtilis ∆bkdAB + LAAD cultures produced a 1.75 times higher yield of KIC (3.37 mmol per L of culture) than the yield of the B. subtilis ∆bkdAB culture. Moreover, we observed a higher amino acid utilization rate and a lower CO2 emission in B. subtilis ∆bkdAB + LAAD cultures compared to that in wild-type B. subtilis cultures. Altogether, this thesis demonstrated a successful microbial fermentation system capable of converting solid organic waste into the non-toxic, value-added products. This system represents an addition to circular bioeconomy, buttressing the 12th goal (responsible consumption and production) of the Sustainable Development Goals (SDGs) and facilitating the net-zero CO2 emission. |
