白蟻窩內細菌相分析及其總菌群與組成菌種降解生質的潛力

Abstract

因為碳水化合物水解酵素是發展第二代生質酒精中重要的一環，所以本文著重在探討白蟻窩內微生物酵素降解生質的潛力。本研究從蟻窩中篩選出可培養的108隻細菌，然後根據酵素測試將其分為18組。根據定序的結果它們全部屬於Bacillus屬。且經由酵素測試，發現這些菌種能夠分泌許多酵素，例如:澱粉酶、羧甲基纖維素酶、青苔澱粉酶、木聚醣酶、果膠酶、質酸酶、脂肪酶、蛋白酶。接著，我們從這些可以培養的菌種中挑選出具有較多酵素活性的菌種Bacillus subtilis (TG 12) 和 Bacillus amyloliquefaciens (TG 50)，以及蟻窩中所有混合的菌種(TG ALL)。利用相思樹未漂漿和已漂漿與25% (w/v) Luria-Bertani borth (LB)培養基和M9培養基為基質培養，測試這三組菌群在上述環境中對羧甲基纖維素酶、外切型纖維素酶、木聚醣酶、β葡糖苷酶的誘導情形。結果顯示，這三組菌群分泌出大部分酵素活性在25% LB培養基中大於在M9培養基中。且TG 12 和TG 50 的酵素活性比TG ALL 顯著。再者，在這些試驗中發現，活性大小依次如: xylanase, Avicelase, CMCase, β-Glucosidase。此外，透過聚合度和還原糖測試它們在25% LB培養基和M9培養基對相思樹未漂漿和已漂漿的降解能力。TG 12、TG 50和TG ALL 培養在25% LB 培養基可以將相思樹未漂漿的LODP (可降解之最低聚合度)降低至原始聚合度(DP) 的79.5 %, 81.1% 和 79.6%；降低相思樹已漂漿的LODP至 79.9 %, 83.5% 和 87.7%。而培養在M9培養基可以降解相思樹未漂漿的LODP至原始聚合度(DP) 的79.5 %、85.8%、83.6.%；降低相思樹已漂漿的LODP至85.0 %、89.7% 、88.3%。

Because carbohydrate hydrolysis enzymes play an important role in bioethanol production, this study aims at the abilities of microorganism derived from termite nest to degrade biomass. 108 culturable bacteria for degradation test were selected, and through the enzyme activity assay they can be classified into 18 groups. After sequencing, all of these species are found to belong to one genus, Bacillus. The enzyme activity assay result shows that many enzymes, such as amylase, CMCase, licheninase, pectinase, xylanase, phytase, lipase and protease, are contained in the bacteria derived from the termite nest. To perform the pulp degradation test, we use three samples of different bacteria configuration to examine various enzyme activities: The sample of all kinds of culturable bacteria derived from termite nest, was coded as TG ALL, Bacillus subtilis and Bacillus amyloliquefaciens were nominated as TG 12 and TG 50. The enzymatic test include CMCase, Avicelase, Xylanase and β-Glucosidase, and the inducing conditions are in a 25% (w/v) Luria-Bertani borth (LB) medium and an M9 medium, respectively. In addition, acacia unbleached and bleached pulp is fed to judge the bleaching effect on the enzyme activity. The results show that enzyme activities are higher in the 25% LB medium than in the M9 medium. Also, TG 12 or TG 50 has higher activities than TG ALL. The ranking of the induced enzyme activity is as follows: xylanase, Avicelase, CMCase, β-Glucosidase. Through the intrinsic viscosity and released reducing sugar tests, we found that all the cultured samples can degrade acacia unbleached pulp DP off to 79.5 %, 81.1% and 79.6%; and degrade acacia bleached pulp DP of to LODP to 79.9 %, 83.5% and 87.7% of LODP in the 25% LB medium. On the other hand, they can degrade acacia unbleached pulp DP off to 79.5 %, 85.8% and 83.6%; and degrade acacia bleached pulp DP of to LODP to 85.0 %, 89.7% and 88.3% of LODP in the M9 medium.