An Xylitol Conversion Process by Fermentation from Pulp Mill Wastes

木質材料的利用過程中，常產生大量的未被充分利用之木質廢棄物，如在製漿操作過程中所產生的木屑。在本研究中，探討利用漿廠的木質廢棄物轉化木糖醇之效能，首先利用硫酸作為前處理水解木質廢棄物，接著以發酵與超過濾做處理。 在水解處理中，使用水、氫氧化鈉、4% 過氧化氫與3%硫酸作為溶液水解廢棄木屑，而以3%硫酸於140oC下水解60分鐘可得最佳之木糖產量12.31 g L-1 (葡萄糖為4.42 g L-1、阿拉伯糖為0.30 g L-1)。再者，使用硫酸水解不同的漿廠廢棄物比較木糖的產生量，其中以澳洲尤加利 (Eucalyptus globules) 木片有最高木糖量為13.35 g L-1，雖然其有最高產率，但使用廢棄木屑較為經濟。廢棄木屑亦有較高的木糖量為12.31 g L-1，其含量較澳洲尤加利木片少。 本研究為確定發酵製程之可行性，利用混糖基質添加氮源以酵母發酵生產木糖醇，試驗菌種獲得於新竹食品工業發展研究所生物資源保存及研究中心，包括Candida boidinii (BCRC 21432)、C. guilliermondii (BCRC 21549)、C. tropicalis (BCRC 20520)、C. utilis (BCRC 20334)與Pichia anomala (BCRC 21359)。其中以C. tropicalis在培養48小時後具最高的生物轉化率 (YP/S) 為 0.79 g g−1。另以尿素與大豆取代酵母抽出物作為氮源，分別發酵96與72小時後，可得YP/S分別為0.6 g g−1 與0.39 g g−1。發酵液以超過濾方式去除發酵產生的蛋白質，使用10000分子量 (MWCO) 超過濾聚醚砜膜(ultrafiltration polyethersulfone membrane )，以2 mg L−1 polydiallyl dimethyl ammonium chloride (PDADAC) 處理膜表面，再以3 kgf/cm2的壓力以掃流式方式過濾，過濾120分鐘後，約85至96%的蛋白質可被去除。 確定發酵製程之可行性後，在以木屑水解液以去毒處理後濃縮作為發酵基質。前處理是以3% (w/v) 硫酸在140oC下水解1小時，並以Ca(OH)2中和，隨後利用活性炭與陰離子交換樹脂移除水解液中的糠醛、5-羥甲基糠醛、酚類物質與醋酸。使用已去毒並濃縮的水解液作為木糖醇生產的發酵基質，比較五種酵母菌對於木糖醇的轉化能力，其中以熱帶假絲酵母有最高的生物轉化率 (YP/S)，於發酵44小時時為0.73 g g−1，另探討廉價發酵培養基的利用可能性，分別添加尿素與大豆粉取代酵母抽出物，C. tropicalis在發酵96小時，其各單位基質的產量分別為0.43 g g−1 與 0.28 g g−1。在發酵液中的木糖可利用分子量為5000超過濾聚醚砜，以2 mg L−1 PDADAC處理固著於膜表面，再以3 kgf/cm2的壓力以掃流式方式過濾，過濾120分鐘後，約82至97%的蛋白質可被去除。在本研究中，證實廉價的發酵培養基可被應用於木質廢棄物轉化為木糖醇的製程。Substantial amounts of underutilized wood wastes are created by chip storage and handling operation in pulp mills. This paper reports a process for xylitol conversion from pulp mill hardwood wastes, starting from pretreated waste wood acid hydrolysate followed by fermentation and ultrafiltration. Using the different solvent to hydrolyze the wood fines, including water, NaOH, 4% H2O2 and 3% H2SO4, the best hydrolysis solvent was 3% H2SO4 with condition was at 60 min with 140oC, the xylose yield was 12.31 g L-1 (glucose was 4.42 g L-1and arabinose was 0.30 g L-1). Compared with the yield of xylose in four materials, the first was the Eucalyptus globules (13.35 g L-1), although the Eucalyptus globules had the greatest output in the four materials, the quantity was small in the pulp mill. The waste wood dust, the second (12.31 g L-1), was the better than Eucalyptus globules, the xylose output of waste wood dust was slightly less than the Eucalyptus globules, but the waste wood dust was great quantity. Batch fermentations for xylitol production using the model mixed sugars media were conducted with conventional a nitrogen source using the strain with optimal xylitol conversion efficiency, conventional nitrogen source was employed using following yeast strains obtained from the Bioresources Collection and Resource Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsin-Chu, Taiwan: Candida boidinii (BCRC 21432), C. guilliermondii (BCRC 21549), C. tropicalis (BCRC 20520), C. utilis (BCRC 20334), Pichia anomala (BCRC 21359), with C. tropicalis exhibiting highest bioconversion yield (YP/S) of 0.79 g g−1 over 48 h. Additional fermentation runs achieved YP/S of 0.6 g g−1 and 0.39 g g−1 after 96 and 72 h with urea and soybean meal, respectively. Xylose in fermentation broth was recovered by ultrafiltration with a cross flow module. With prior application of 2 mg L−1 polydiallyldimethylammonium chloride (PDADAC) on the membrane surface, protein rejections increased from 85 to 96 %. Fermentation hydrolysate, pretreatment started with 140 oC, 3% (w/v) H2SO4 acid hydrolysis for 1 hour, neutralization by Ca(OH)2, followed by sequential removals by furfural, 5-hydroxylmethyl furfural, total phenols and acetic acid with powdered activated carbon and cationic ion exchange columns. Using detoxified and concentrated hydrolysate, batch fermentations for xylitol production were conducted with five yeast strains. Among fermentation runs of the strains, C. tropicalis exhibited highest bioconversion yield (YP/S) of 0.73 g g−1 over 44 h. To investigate feasibility using low-cost fermentation media, additional C. tropicalis fermentation runs achieved YP/S of 0.43 g g−1 and 0.28 g g−1 after 96 h with urea and soybean meal, respectively. Xylose in fermentation broth was recovered by a 5000 molecular weight cutoff (MWCO) ultrafiltration polyethersulfone membrane in a cross flow module with 3 kg/cm2 trans membrane pressure. With prior application of 2 mg L−1 PDADAC on membrane surface, protein rejections increased from 87 to 100 % within 120 min. The present study demonstrates xylitol, a value added derivative, can be effectively converted from hardwood wastes using low-cost fermentation media.Index Page 摘要-----------------------------------------------------I Abstract------------------------------------------------III Index-----------------------------------------------------V Table index---------------------------------------------VII Figure index---------------------------------------------IX I Introduction--------------------------------------------1 II Literature review--------------------------------------3 2.1 Natural occurrences, properties and application of Xylitol-----------------------------------------------3 2.2 Processes for xylitol production----------------------5 2.2.1 Solid-liquid extraction-----------------------------6 2.2.2 Chemical synthesis----------------------------------7 2.3 Microbial production of xylitol-----------------------8 2.3.1 Effect of xylose concentration on xylitol production-----------------------------------------10 2.3.2 Effect of other sugar on the production of xylitol by yeast-------------------------------------------10 2.3.3 Effect of culture condition------------------------10 2.3.4 Production of xylitol from hemicellulose hydrolysate- -------------------------------------------------- 11 III Materials and methods-------------------------------13 3.1 Research Framework-----------------------------------13 3.2 Preparation of lignocellulosic hydrolysate---------- 15 3.2 Detoxification of hydrolysate------------------------16 3.3 Microorganisms and pre-culture-----------------------16 3.4 Media and fermentation------------------------------ 17 3.5 Ultrafiltration------------------------------------- 18 3.6 Analytical methods-----------------------------------19 IV Results and discussion--------------------------------19 4.1 Pretreatment of lignocellulosic materials------------19 4.1.1 Water hydrolysis with wood fines ------------------19 4.1.2 Sodium hydroxide hydrolysis with wood fines ---20 4.1.3 Hydrogen peroxide hydrolysis with wood fines ------20 4.1.4 Sulfuric acid hydrolysis with four materials ------22 4.2 Fermentation of simulated mixed sugar ---------------26 4.2.1 Effect of the yeast strain on fermentation --------26 4.2.2 Effect of the nitrogen sources on fermentation ----27 4.2.3 Ultrafiltration -----------------------------------29 4.3 Fermentation of detoxified hydrolysates -------------30 4.3.1 Effect of detoxification on hydrolysate composition-- ---------------------------------------------------30 4.3.2 Effect of the yeast strain on fermentation---------32 4.3.3 Effect of the nitrogen sources on C. tropicalis fermentation---------------------------------------35 4.3.4 Ultrafiltration------------------------------------36 V Conclusion---------------------------------------------38 VI Reference---------------------------------------------39 Appendix-------------------------------------------------4