The Development of Production Technology on Biobutanol from Monostroma Nitidum

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Project title

Code/計畫編號

MOST103-ET-E019-001-ET

Translated Name/計畫中文名

青海菜生質丁醇生產技術研發

Project Coordinator/計畫主持人

Chorng-Liang Pan

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=8175261

Year

2014

Start date/計畫起

01-01-2014

Expected Completion/計畫迄

01-12-2014

Bugetid/研究經費

615千元

ResearchField/研究領域

能源工程
生物技術(理)

本研究計畫之目的為研發自綠藻青海菜 (Monostroma nitidum) 生產生質丁醇的發酵技術。主要工作包括將青海菜或青海菜熱萃多醣以熱酸、市售纖維素酶、及菌株 Pseudomonas vesicularis MA103 (MA103) 與Aeromonas salmonicida MAEF108 (MAEF108) 所產多重粗酵素液水解，以批式與饋料批式發酵探討提升丁醇產率之生產條件。去鹽與未去鹽5% (w/v) 之青海菜熱酸水解液中之還原醣量分別為0.25 g與1.5 g及鹽濃度為1.46 mg/mL與1.75 mg/mL。熱酸水解反應之基質 (5 g) 分3次逐次添加與1次添加，5% (w/v) 青海菜水解液100 mL中分別產生1.92 g與 1.93 g的總醣及1.33 g與1.28 g的還原醣，顯示一次添加基質者有較佳的水解效果，且生產成本較低。100 mL 5% (w/v)青海菜水解液中加入市售纖維素酶760 U與4,560 U於pH 4.5、37oC下水解1天，可分別產生2.39 g與2.97 g的總醣、1.88 g與 2.04 g的還原醣，顯示4,560 U市售纖維素酶可獲得較高的水解效率，但因成本偏高，後續實驗仍以添加760 U市售纖維素酶進行；分別以粉末、新鮮與去鹽青海菜水解液誘導MA103與MAEF108所產多重粗酵素液生成，結果顯示在第3天時去鹽青海菜水解液誘導者呈現較高之洋菜酶與澱粉酶酵素活性。Clostridium acetobutylicum BCRC10639與C. beijerinckii BCRC17950耐鹽性產丁醇適應菌株的篩選試驗獲得2株具有4.5% 食鹽耐受能力之衍生菌株，命名為C. acetobutylicum BCRC10639- ST (salt tolerant)與C. beijerinckii BCRC17950-ST (salt tolerant)。以濃度30% 與5% 之丁醇溶液進行氣提效率測試時之回收率分別為34% 與33%，而經由殘留丁醇濃度換算可知過程中丁醇散失率分別為54% 與57%。以未去鹽與去鹽青海菜為原料，接種C. acetobutylicum BCRC10639與C. beijerinckii BCRC17950以及耐鹽性的 C. acetobutylicum BCRC10639-ST 與 C. beijerinckii BCRC17950-ST在 37oC下厭氧批式發酵5天，結果顯示未去鹽之青海菜水解液，2株耐鹽性菌株所得之丁醇產率為3.4% (3.4 g/100 mL)。以去鹽者酵素水解後為基質接種 C. acetobutylicum BCRC10639與C. beijerinckii BCRC17950以及耐鹽性的C. acetobutylicum BCRC10639-ST 與 C. beijerinckii BCRC17950-ST菌株所得丁醇產率分別為4.4%與6.7%。以去鹽青海菜為原料配合氣提裝置進行5天丁醇批式發酵，產率為7.01%。去鹽與未去鹽青海菜水解液以兩株耐鹽菌在37oC下厭氧饋料批式發酵5天，可以得知未去鹽青海菜水解液在發酵第1天時有最大丁醇產率0.67%，而去鹽青海菜水解液則在發酵第2天時有最大丁醇產率5.55%，此時丁醇濃度為0.416%，第5天丁醇濃度上升至0.476%，而丁醇產率則下降至4.23%。 The objective of this study is to develop fermentation techniques for producing bio-butanol from green algae Monostroma nitidum. The PS digesting enzymes, from strains MA103 and MAEF108, degrade M. nitidum to obtain fermentable simple sugars (FSS), which can be further metabolized and fermented to butanol by clostridia. The result indicated total sugars (TS) are 1.92 and 1.93 g, and RS are 1.33 and 1.28 g, respectively. The group which added M. nitidum in once had similar results (lower production cost). Cellulases (760 or 4,560 U) is reacted with M. nitidum hydrolysate at pH 4.5 and 37oC for 24 h, results showed TS were 2.39 and 2.97 g, and RS were 1.88 and 2.04 g, respectively. The 4,560 U cellulase group has better yield, but higher cost. Therefore, 760 U cellulase was used for following experiment. Multiple crude enzymes from strains P. vesicularis MA103 (MA103) and A. salmonicida MAEF108 (MAEF108) were induced by 3 kinds M. nitidum hydrolysate: dry powder, fresh, and desalting. Result indicated that strains MAEF108 and MA 103 had better enzyme activities at the 3rd day when the desalted M. nitidum used as inducer. Adaptation to higher salt conc. on strains Clostridium acetobutylicum BCRC10639 and, C. beijerinckii BCRC17950 were conducted with 4.5% salt concentration, and 2 salt tolerant strains obtained, which named BCRC10639-ST (salt tolerant) and BCRC17950-ST. Gas-stripping system was tested for its recovery rate with 30% and 5% butanol soln., both recovery rate is about 34%. Butanol yields of hydrolysate of desalted M. nitidum fermented by BCRC10639/17950 and BCRC10639-ST/17950-ST are 4.4% and 6.7%, respectively. After gas striping technique is applied, the butanol yield increased to 7.01% as desalted M. nitidum used. In the fed-betch fermentation with desalted M. nitidum hydrolysate, the maximun butanol yield fermented by BCRC10639-ST/17950-ST is 5.55% at day 2 (butanol conc., 0.42%) and the butanol concentration reached 0.48% at day 5.

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The Development of Production Technology on Biobutanol from Monostroma Nitidum

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