氣霧式生物反應器之改良及應用於台灣金線連與白芷之生產

Abstract

本研究之目的在於分析由不同超音波換能器自含糖與不含糖之MS (Murashige and Skoog, 1962)液態培養基激發產生營養液氣霧的化學組成(碳水化合物及各種無機元素)，以評估氣霧式生物反應器的效率，並利用此營養液霧化生物反應器(nutrient mist bioreactor, NMB)培育台灣金線連(Anoectochilus formosanus Hayata)種苗和白芷〔Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook.〕微塊莖。 未添加蔗糖的MS培養基在滅菌前、後之pH值皆為5.2，若是2.8 MHz- 17.5W處理組樣本的pH值則為5.4。存在於2.8 MHz- 17.5W處理組樣本內之大量元素(NH4+-N, NO3－-N, P, K, Mg, Ca 和S)與微量元素(Na, Fe, Mn, Zn, B, I, Mo和Co)含量，也與滅菌後的MS培養基無顯著差異。添加30 g•L-1蔗糖的MS培養基在滅菌前的pH值為5.2(對照組A)，但滅菌後(對照組B)降低至4.8。以2.8 MHz超音波換能器配合17.5W輸入功率為最理想的霧化條件，因為該處理組樣本的pH值(4.6 ± 0.1)與滅菌後的MS培養基無明顯差異，並且葡萄糖(1.7 g•L-1)、蔗糖(25 g•L-1)、可溶性碳水化合物(29.6 g•L-1)、大量元素(NH4+-N, NO3－-N, P, K, Mg, Ca 和S)與微量元素(Na, Fe, Mn, Zn, B, I, Mo和Co)含量，也與滅菌後的MS培養基無顯著差異，但果糖含量(2.9 g•L-1)則顯著高於所有的處理組。此外，當生物反應器中的MS培養基換成B-5或WPM培養基，並且使用2.8 MHz- 17.5W霧化條件時，其激發產生的營養液霧氣NH4+-N和NO3－-N含量，也分別與滅菌後的B-5或WPM培養基無顯著差異。 改良的NMB系統主要構件包含伺服馬達、氣體濃度感測器、溫度感測器、導氣管、栽培架、霧化器、蠕動幫浦、控制器、電腦及液晶顯示器。當超音波換能器霧化時間設定為80 sec並配合3 L•min-1氣體流量和6 rpm栽培架轉速時，水試紙濕潤狀況較均勻，故為最適宜的操作條件。新鮮養液補充次數在第8次後，霧化器內之培養液pH值(4.7)、可溶性固形物含量(3.4 %)及NH4＋-N含量(306.0 mg•L-1)皆與滅菌後的MS培養基(對照組，4.9、3.3 %及291.9 mg•L-1)無顯著差異。然而，霧化器內培養液的NO3－-N含量(643.7 mg•L-1)則顯著高於對照組(613.0 mg•L-1)。 應用改良的NMB系統分別培育台灣金線連植株或白芷微塊莖60天後，其gastrodin (626.1 μg•g-1)或imperatorin (387.5 μg•g-1)含量皆與培養於傳統蘭花瓶、聚碳酸酯容器或溫室者無明顯差異。台灣金線連的平均莖徑(3.7 mm)和單株鮮重(2.6 g)，以及白芷微塊莖的體積(1.2 cm3)、鮮重(1.2 g)和乾物重(0.12 g)也與其他處理組無顯著差異。當營養液氣霧供應週期為每日8次時，台灣金線連或白芷的生長量與每日供應16次的處理組無顯著差異；然而，前者微塊莖中的imperatorin含量(563.3 μg•g-1)則高於後者(387.5 μg•g-1)。此外，本試驗也分別估算單位面積生產台灣金線連與白芷之變動成本。

The objectives of this study was to evaluate the efficiency of ultrasonic transducers on generating nutrient mist by analyzing the chemical constituents (carbohydrates and inorganic elements) of the condensed mists generated by ultrasonic transducers from liquid sucrose-supplemented and sugarless MS (Murashige and Skoog, 1962) media. Furthermore, develop a useful nutrient mist bioreactor (NMB) for growing Anoectochilus formosanus Hayata plantlets and Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. microtubers. The pH value of sugarless MS medium before or after autoclaving was at 5.2. The samples average pH value from 2.8 MHz- 17.5W treatment was 5.4. The contents of macro-elements (NH4+-N, NO3－-N, P, K, Mg, Ca and S) and micro-elements (Na, Fe, Mn, Zn, B, I, Mo and Co) in 2.8 MHz- 17.5W sample were similar to those in the MS medium after autoclaving. The pH value of sucrose-supplemented (30 g•L-1) MS medium before autoclaving was 5.2 (control A), while reduced to 4.8 after autoclaving (control B). The ultrasonic transducer oscillation frequency with an input power of 2.8 MHz and at 17.5W was the optimum condition since the contents of glucose (1.7 g•L-1), sucrose (25 g•L-1), total soluble carbohydrate (29.6 g•L-1), macro-elements (NH4+-N, NO3－-N, P, K, Mg, Ca and S), and micro-elements (Na, Fe, Mn, Zn, B, I, Mo and Co) value were similar to those in the MS medium after autoclaving. However, the content of fructose (2.9 g•L-1) in 2.8 MHz- 17.5W sample was significantly larger than those in other treatments. Furthermore, when MS medium was replaced by Gamborg''s medium (B-5) or woody plant medium (WPM) in this bioreactor at the same settings (2.8 MHz- 17.5W) of transducer, the NH4+-N and NO3－-N contents in nutrient mist generated were also similar to those in B5 or WPM after autoclaving, respectively. The major configurations of an improvement NMB system contained served motor, air concentration sensor, temperature sensor, airway, cultured shelf, atomizer, tubing pump, controller, computer, and LCD monitor. When the mist generated by ultrasonic transducer for 80 sec combined with 3 L•min-1 air flow, the cultural shelf rotated at 6 rpm was the optimum condition, since wetted the water-sensitive papers uniform. The frequency of fresh medium enriched after eight times, the pH value (4.7), soluble solid content (3.4 %) and NH4＋-N content (306.0 mg•L-1) of liquid medium in NMB were not significantly different from MS medium after autoclaving (control, 4.9, 3.3 % and 291.9 mg•L-1). However, the NO3－-N content (643.7 mg•L-1) of liquid medium in NMB was significantly higher than control treatment (613.0 mg•L-1). The gastrodin content (626.1 μg•g-1) of A. formosanus Hayata plantlets or imperatorin content (387.5 μg•g-1) of A. dahurica microtubers cultured in the improvement NMB system for 60 days was the same as those from cultured in traditional flask, polycarbonate vessel or greenhouse. As well as the average stem diameter (3.7 mm) and fresh weight (2.6 g) per plantlet or the size (1.2 cm3), fresh weight (1.2 g) and dry weight (0.12 g) per microtuber cultured in the NMB were also not significantly different from those cultured in flask or greenhouse. On the plantlet or microtuber biomass cultured in the NMB supplied 8 times mist per day was similar to those cultured in NMB supply of 16 times mist per day. However, average imperatorin content in microtuber cultured in the former (563.3 μg•g-1) was higher than that cultured in the later (387.5 μg•g-1). Furthermore, the variable production cost per unit area for A. formosanus Hayata or A. dahurica was estimated, respectively.