本研究利用高溫澱粉水解策略提升二階段產氫效率。此系統主要分為兩階段，先利用微生物法或酵素法進行澱粉前處理，將澱粉轉化成小分子的醣類，以便產氫菌利用，藉此提高暗醱酵生物產氫效率。
本研究首先以微生物進行澱粉水解，主要是利用高溫菌Caldimonas taiwanensis On1以sequencing batch reactor (SBR)操作策略進行澱粉水解，在pH 7、曝氣速率 1 vvm，置換體積90%時，有最佳的澱粉水解效能，還原糖最大產量 13.94 g reducing sugar/L，還原糖最大產生速率 0.92 g R.S/h/L，澱粉最大水解速率為 1.86 g starch/h/L，並且在經過4個cycle的處理後，總共可以將216 g的澱粉水解，並且得到100.4 g還原糖。接著，以Ca. taiwanensis On1澱粉水解產物當基質，提供Clostridium butyricum CGS2進行暗醱酵連續流產氫。當hydraulic retention time (HRT)從12 h調降至2 h時，產氫速率由0.5 L/h/L提升至1.5 L/h/L，比產氫速率由250提升至534 ml H2/g volatile suspended solids/h，氫氣產率由2.03 下降至1.50 mol H2/mol glucose (12.52~9.19 mmol H2/g starch)，氫氣濃度維持在50~60%，菌體濃度維持在 2~3 g VSS/L，pH控制在5.8~6.5。
接著，本研究亦利用基因重組菌Escherichia. coli BL21 (DE3)/pEAmy3063大量表達澱粉酶，並利用其進行澱粉之酵素水解。先以response surface methodology (RSM) 進行澱粉酶環境因子之最佳化，在pH 6.70 和溫度 54.2℃時，有最佳活性 3.80 U/ml，並利用Michaelis-Menten (M-M) model探討動力學參數，可以得到vmax為5.20 U/ml， Km值為5.18 g/L。使用celite為載體，將澱粉酶以共價鍵結的方法進行酵素固定化，在pH 7.0，澱粉酶濃度 50 mg/ml，固定化時間為2 h時，可得到最佳酵素固定化量為 179.86 mg amylase/g celite。並且利用RSM進行celite-amylae環境因子探討，在pH 6.50 和溫度 53.6℃時，有最佳活性 1.24 U/ml，其M-M動力學模式參數分別為vmax=3.04 U/ml、Km=6.79 g/L。以酵素法水解產物進行Cl. butyricum CGS2和Cl. pasteurianum CH4批次產氫實驗。Cl. butyricum CGS2以cassva starch水解產物為基質時，有最大產氫速率 124 ml/h/L，而Cl. pasteurianum CH4以soluble starch水解產物為基質時，有最大的產氫量與產率分別達301 ml和9.95 mmol/g COD。

Biohydrogen production from starch using a two-stage thermophillic hydrolysis/drak H2 fermentation strategy was developed. In the first stage, starch was hydrolyzed by a starch-hydrolytic strain Caldimonas taiwanensis On1 or by amylase produced from recombinant E. coli BL21 (DE3)/pEAmy3063. The recombinant amylase was also immobilized on celite to examine for the feasibility of repeated uses of the enzyme. In the second stage, a pure H2-producing strain Clostridium butyricum CGS2 was used to produce H2 from raw or hydrolyzed starch via dark fermentation. Both batch and continuous fermentative H2 production cultures were employed.
Starch hydrolysis with Ca. taiwanensis On1 was operated using sequencing batch reactor (SBR). The optimal conditions of the SBR were pH, 7.0; aeration rate, 1 vvm; temperature, 55℃. The average reducing sugar production, the sugar production rate and the highest starch hydrolysis rate were 13.94 g R.S./L, 0.92 g R.S./h/L and 1.86 g S/h/L, respectively. After 4 cycles in SBR operation, the maximum amount of starch hydrolyzed and reducing sugar produced were 216 g and 100.4 g, respectively.
Response surface methodology (RSM) with central composite design (CCD) was used to optimize pH and temperature leading to the best amylase activity. The predicted maximum activity of the amylase produced form E. coli BL21 (DE3)/pEAmy3063 was 3.80 U/ml at pH 6.70 and 54.2℃. The value of vmax and Km estimated from simulation of experimental data was 5.20 U/ml and 5.18 g/L, respectively. The optimal conditions of immobilized celite-amylase were pH, 7.0; amylase concentration, 50 mg/ml; immobilization time, 2 h. The maximum amount of immobilized amylase was 179.86 mg amylase/g celite. The optimal environmental factors leading to a predicted maximum celite-amylase activity of 1.24 U/ml was pH 6.50 and 53.6℃. The value of vmax and Km was 3.04 U/ml and 6.79 g/L, respectively.
In the batch hydrogen-producing culture, the maximum hydrogen production rate for Clostridium butyricum CGS2 was nearly 124 ml/h/L while using hydrolysate of cassava starch as the substrate. Despite a lower H2 production rate, Cl. pasteurianum CH4 had a higher maximum hydrogen production (Hmax = 301 ml) and hydrogen yield (YH2=9.95 mmol/g COD) when using hydrolysate of soluble starch as the substrate.
For continuous biohydrogen-producing culture, the operation HRT was decreased from 12 to 2 h using hydrolyzed starch (at a total sugar concentration of 26 g/L) as the feeding substrate. The hydrogen content in biogas stably reached at around 50–60%. The hydrogen production rate increased from 0.5 to 1.5 L/h/L while the HRT decreased from 12 to 2 h. The biomass concentration in the reactor kept within the range of 2 to 3 g VSS/L, while pH varied between 5.8 and 6.5. Meanwhile, the specific hydrogen production rate increased from 250 to 534 ml H2/g VSS/h as HRT decreased from 12 to 2 h, whereas the hydrogen yield decreased slightly from 2.03 to 1.50 mol H2 /mol glucose (i.e., 12.52–9.19 mmol H2/g starch).

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