利用白蟻腸內共生菌Clostridium xylanolyticum分解木質纖維素

Abstract

白蟻腸道內的共生微生物能協助分解木質纖維素，使纖維素和半纖維素降解生成短鏈醣類，本研究利用白蟻腸道細菌Clostridium xylanolyticum進行木質纖維素的分解，首先經剛果紅染色法確認其具有分解羧甲基纖維素鈉與木聚醣的能力，將其培養於Modified PYG medium中可獲得甲酸鹽、醋酸鹽與酒精三種主要醱酵產物，其中醋酸鹽的累積會造成pH值下降，當環境的pH值低於5.0時有明顯的生長抑制現象。建立此菌株的生長曲線後發現0-12小時為遲滯期，12-30小時為對數生長期，30小時以後為靜止期，菌株的生代時間為2.4小時，最佳的接種時機於24-30小時之間。 使用葡萄糖、纖維雙糖、羧甲基纖維素鈉、微晶纖維素Avicel®、木糖與木聚醣進行基質測試，藉此評估菌株分解各種纖維素與半纖維素的能力，結果顯示其對於木聚醣的分解具有較大的優勢，因此選用木聚醣基質進行菌種馴化以提升菌種的分解能力。在菌種馴化的過程中，菌株的木聚醣的分解能力與酸耐受度逐漸提升，在後續基質醱酵的實驗中採用第一次繼代培養後的菌種，因其已具備相當優秀的木聚醣分解能力並能在接種後一天內快速進行醱酵反應。 基質醱酵實驗利用一年生麻竹為天然基質，並在培養過程中進行pH值控制，在培養期的前24小時，菌體迅速消耗醣類基質並產出大量的酸醇產物、氫氣及二氧化碳，麻竹基質中大部分的半纖維素被菌體分解利用殆盡，最後共產出醋酸鹽4.85 g/L、酒精0.14 g/L、氫氣99.3 mL與二氧化碳179.6 mL，其中醋酸鹽可作為厭氧醱酵生產甲烷的基質，氫氣為氣態燃料，酒精則可作為液態燃料使用。

It was found that the symbiotic microorganisms in termite gut can digest lignocellulose, i.e., to degrade the cellulose and hemicellulose into short-chain oligosaccharides. In this study, Clostridium xylanolyticum, a symbiotic bacterium in termite gut has been used to degrade the lignocellulose. At the beginning, Congo red dye assay was applied to identify its capability in degradation of sodium carboxymethyl cellulose (CMC-Na) and xylan, and then cultivated with the modified peptone–yeast extract-glucose (PYG) medium. The major fermentation products are formate, acetate and ethanol. The accumulation of acetate will result in the decrease of pH, and a significant antiproliferative effect will occur when the ambient pH is lower than 5.0. The growth curve for this particular bacterium showed a 12 hrs lag phase, an 18 hrs exponential growth phase, and followed consequently the stationary phase. The mean generation time was found to be 2.4 hrs, and the optimal inoculation time was between 24-30 hrs. To evaluate its degradation ability about the cellulose and hemicellulose, experiments using different substrates such as glucose, cellobiose, sodium carboxymethyl cellulose, microcrystalline cellulose Avicel® PH-101, xylose, and xylan were conducted. The results showed Clostridium xylanolyticum had preference in degradation of xylan. Therefore, xylan was then selected as the substrate for acclimation of the inoculum. During acclimation, the ability of xylan degradation and acid resistance were significantly improved. In follow-up substrate fermentation experiment, the first subculture was used as the inoculum for its superiority in xylan degradation and could be reacted rapidly after one day of inoculation. In fermentation experiment of the annual Ma Bamboo, its sugar content was rapidly consumed at beginning 24 hrs, and large amount of acids, alcohol, hydrogen and carbon dioxide were produced with pH under control. Most hemicellulose in Ma Bamboo were almost completely used in this experiment, and 4.85 g/L of acetate, 0.14 g/L of alcohol, 99.3 mL of hydrogen and 179.6 mL of carbon dioxide were produced. Among these products, the acetate can be used as the substrate for methane fermentation, while hydrogen and alcohol can be used as the gaseous and liquid fuel, respectively.