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研究生:吳耿銘
研究生(外文):Keng-Ming Wu
論文名稱:微藻培養基質之微波滅菌系統研發
論文名稱(外文):Development of a microwave sterilization system using for the culture medium of microalgae
指導教授:尤瓊琦
指導教授(外文):Chung-Chyi Yu
口試委員:黃振文洪滉祐
口試委員(外文):Jenn-Wen HuangHuaang-Youh Hurng
口試日期:2015-07-21
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物產業機電工程學系所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:126
中文關鍵詞:微藻微波微藻培養基質滅菌微波滅菌
外文關鍵詞:microalgaemicrowavemicroalgae culture medium sterilizationmicrowave sterilization
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  • 下載下載:33
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本研究旨在建立微藻培養基質之微波滅菌系統,用以改善傳統在微藻培養基質滅菌方式使用高壓滅菌與次氯酸鈉所造成費時與需去除水中殘餘化學物質等問題。試驗首先進行連續式微波滅菌可行性試驗,使用等鞭金藻(Isochrysis galbana, CCAP 927/1)、紫球藻(Porphyridium cruentum, CCAP 1380/3)、擬球藻(Nannochloropsis oculata, CCAP 849/1) 、周式扁藻(Tetraselmis chui, CCAP 8/6)及角毛藻(Chaetoceros muelleri, CCAP 1010/3)等5種微藻藻種混合溶液作為培養基質試驗樣本,並在額定功率530 W與微波頻率2450 MHz之家用型微波爐中設置玻璃蛇管,且使用蠕動幫浦進行控制進出料流率39.3、56.6、71.3、87.3、109.7、178.7、241.3及321.3 ml/min對樣本進行處理,藉以測試經微波處理後之培養基質樣本是否與傳統高壓滅菌處理方式具有相同結果,進而尋找滅菌指標藻種,選用作為後續試驗樣本。試驗結果獲致藻水混合溶液,在處理流率178.7ml/min以下,即其藻水溶液微波停留時間23.5 s以上時,其固態微藻培養基之平板培養與曝氣培養,皆顯示其與以溫度121℃與滅菌時間30 min之高壓滅菌釜有相同之效果,且獲致擬球藻(Nannochloropsis oculata)在5種微藻藻種中,較能抵抗微波之照射,因此未來試驗將以擬球藻作為微藻之試驗樣本。
其次為改善家用型微波爐之多模共振腔體所造成之加熱區域限制,本研究設計開發2種共振腔體長度之TM010模態之單模圓柱形共振腔體,分別是共振腔長度為0.129 m之微波腔體a與共振腔長度為0.172 m之微波腔體b,並探討樣本經微波腔體內不同處理管外(內)徑8(6)、10(8)及12(10) mm與處理流率20.6、82.3、143.9、205.6、267.3及328.9ml/min處理後之影響。試驗是以擬球藻(Nannochloropsis oculata)溶液作為試驗樣本進行連續式單模共振微波系統滅菌藻水試驗,經微波處理後之試驗樣本將分別以固態微藻培養基與固態菌落培養基進行平板培養,並分別於培養96 hr與14 hr後觀察微藻生長情形與量測菌落數,且處理後之試驗樣本亦置於震盪培養箱中培養96 hr,每24 hr量測其細胞密度、吸光值及葉綠素螢光,作為滅菌效果評斷之依據。連續式單模共振微波系統滅菌藻水試驗結果獲致在微波腔體b與處理管外徑12mm操作條件下,具有最好之平均加熱功率341.6±22.1 W,且具有能有效殺死擬球藻與其溶液中之雜菌之最大處理流率為205.6 ml/min,其相對應藻水溶液微波停留時間為3.9 s。
試驗為進一步檢測微波系統之滅菌效果,選取對微波耐受度較高之枯草芽孢桿菌(Bacillus subtilis, 01)菌液作為試驗樣本,並以前一試驗獲致有最好平均加熱功率與滅菌效果之微波腔體b、處理管外徑12mm及處理流率為20.6、82.3、143.9、205.6、267.3及328.9ml/min作為操作參數,其相對應菌液溶液微波停留時間分別為39.3、9.9、5.6、3.9、3.0及2.5 s,進行連續式單模共振微波系統之滅菌菌液試驗。試驗亦透過1~4次重覆滅菌,增加其菌液微波停留時間。且分別將經處理後之菌液樣本接種至固態菌落培養基進行平板培養,並於接種後14 hr量測菌落數,用以獲致各處理流率之滅菌效果。連續式單模共振微波系統之單次滅菌菌液試驗結果中,獲致在微波腔體b與處理管外徑12mm之各處理流率組仍皆未能有效達到完全滅菌效果,其枯草芽孢桿菌菌落數與處理流率呈一正比關係,在處理流率20.6ml/min,相對應菌液溶液微波停留時間39.3s組,獲致最低菌落數,顯示其較擬球藻有較大耐受微波之能力,試驗欲達完全滅菌效果,其微波處理停留時間亦需加長,因此於重複滅菌試驗中獲致,在微波腔體b、處理管外徑12mm及處理流率20.6ml/min之操作條件下,重複滅菌次數4次與相對應菌液微波停留時間157.2 s時,則能完全殺死枯草芽孢桿菌。
試驗最終以連續式單模共振微波系統之滅菌藻水試驗獲致能有效滅菌之微波操作參數,其是微波腔體b、處理管外徑12 mm及處理流率20.6、82.3、143.9及205.6 ml/min,作為連續式單模共振微波系統之海水滅菌試驗之微波操作參數進行試驗,並將經微波處理過後之海水培養基質,接種角毛藻(Chaetoceros muelleri, CCAP 1010/3)於1公升錐形瓶中進行曝氣(1 l/min)培養96 hr,且每24hr量測其細胞密度與吸光值。試驗獲致微波腔體b與處理管外徑12mm操作條件下,具有最大處理流率205.6ml/min與最短微波停留時間3.9 s,其角毛藻培養結果並未出現感染之現象,且角毛藻於微波處理之海水培養基質中成長情形略優於經高壓滅菌處理之海水培養基質中;本研究獲致以微波滅菌系統進行微藻培養基質滅菌,能提供相較於傳統使用高壓滅菌與次氯酸鈉等滅菌方式,較高效率且無化學汙染之滅菌方式。


The purpose of this study was to develop a microwave sterilization system used to improve time consumption and resolve the chemical residues problems of microalgae culture medium treated by the traditional autoclave and sodium hypochlorite methods, respectively. The feasibility experiment of continuous microwave sterilization using microalgae suspension combined with five species of microalgae, Isochrysis galbana (CCAP 927/1), Porphyridium purpureum (CCAP 1380/3), Nannochloropsis oculata (CCAP 849/1), Tetraselmis chui (CCAP 8/6) and Chaetoceros muelleri (CCAP 1010/3) was first conducted. The samples were treated through the spiral tube equipped in the domestic microwave oven under the rated power 530 W and frequency 2450 MHz. Five sets of feeding flow rate 39.3, 56.6, 71.3, 87.3, 109.7, 178.7, 241.3 and 321.3 ml/min were chosen and controlled by a peristaltic pump. The experimental results would compare to the case sterilized by autoclave and used to find the indicator microalgae for the case in the following experiments. The results showed that samples inoculated on the microalgae solid medium and cultured with aeration after treating by microwave as the flow rates smaller than or equal to178.7 ml/min, with the corresponding resident time lager than or equal to 23.5 s, same sterilization phenomenon was obtained with the samples treated by autoclave with sterilization temperature at 121℃ and sterilization time for 30 min. The experiments also obtained that Nannochloropsis oculata (CCAP 849/1) held stronger ablilty to endure microwave in these five microalgae species, and it was selected as the testing microalgae in the following experiment.
Secondly, in order to improve the uneven heating problems caused by the domestic microwave oven which is a multi-mode resonator, two lengths of the resonator chamber based on the design regulation TM010 of the single-mode circular resonator were chosen, and that according to the calculated radius of the resonator, resonator a and b were assigned to the resonator with length 0.129 and 0.172m, respectively. The effects of microwave on the experimental samples by using different sizes of straight tube with outer (inner) diameter 8 (6), 10 (8), and 12 (10) mm, and feeding flow rates 20.63, 82.3, 143.9, 205.6, 267.3 and 328.9 ml/min were studied. The newly developed microwave system with single-mode resonator would sterilize microalgae suspension Nannochloropsis oculata (CCAP 849/1) continuously. The experimental samples treated by microwave would then inoculate on the microalgae solid medium and the colonies solid medium, and the growing phenomena on the microalgae and colonies solid medium were observed after cultivation of 96 hr and 14 hr, respectively. The experimental samples were also cultured in an incubator for 96 hr, and the cell density, absorbance and chlorophyll fluorescence were measured every 24 hr, which are used as the parameters to further evaluate the sterilization effectiveness. After the analysis of the experiment results,it showed that the case using microwave resonator b and 12mm-outer-diameter tube with the average heating power 341.6±22.1 W, flow rate 205.6 ml/min and resident time 3.9 s was the optimal case in this test, which could kill the cells of Nannochloropsis oculata (CCAP 849/1) and bacteria completely.
Thirdly, the sterilization ability obtained in the previous experiment of the newly developed microwave system would be further justified, the system combined with resonator b and 12mm-outer-diameter tube was selected to sterilize the bacteria suspension Bacillus subtilis (01) continuously. Six flow rates conditions 20.6, 82.3, 143.9, 205.6, 267.3 and 328.9 ml/min were used, and the corresponding resident times were 39.3, 9.9, 5.6, 3.9, 3.0 and 2.5 s, respectively. Moreover, each set of samples was treated 1~4 cycles respectively to increase the microwave resident time on the bacteria suspension. The experimental samples after treated by microwave would then inoculate on the colonies solid medium. The number of colonies growing was calculated after cultivation 14 hr and which was used to further evaluate the sterilization effectiveness. After the analysis of the experiment results, it showed that each operating condition could not kill Bacillus subtilis (01) completely as the sample only treated one cycle. The number of colonies growing obtained are proportional to the flow rate, and the case with flow rate 20.6 ml/min and resident time 39.3 s demonstrated the least colonies growing phenomena observed on bacteria solid medium. The experiment results also showed that Bacillus subtilis (01) was able to tolerate microwave more than Nannochloropsis oculata, so in order to sterilize Bacillus subtilis (01) completely, the resident time of bacteria suspension would have to be increased. The case of sterilization cycle repeated 4 times withflow rate 20.6 ml/min and total resident time 157.2 s then could kill Bacillus subtilis (01) completely.
Finally, the microwave system newly developed using resonator b and 12mm-outer-diameter tube under the flow rates 20.6, 82.3, 143.9 and 205.6 ml/min and the corresponding resident times 39.3, 9.9, 5.6 and 3.9 s as the operating conditions that could sterilize microalgae medium completely obtained from the previous experiment was then used to contiously sterilize seawater for the proved study. The seawater treated by microwave would then be inoculated Chaetoceros muelleri (CCAP 1010/3), and cultured in the Erlenmeyer flasks (1 l) with aeration rate 1 l/min for 96 hr. The cell density and absorbance of Chaetoceros muelleri (CCAP 1010/3) were measured every 24 hr. The experiment results obtained that the cases using resonator b and 12mm-outer-diameter tube with flow rate smaller than or equal to 205.6ml/min and resident time lager than or equal to 3.9 s were no contamination occurring during the culture period. The experiment results also showed that the growing parameters such as cell density and absorbance of Chaetoceros muelleri (CCAP 1010/3) cultured in the seawater treated by microwave were better than the one treated by autoclave. This research obtained that using microwave to sterilize the microalgae culture medium could provide higher efficiency and no chemical residues left as compared to the traditional autoclave and sodium hypochlorite methods.


謝誌 ..……………………………………………………………………I
摘要 ..…………………………………………………………………...II
Abstrac ...…………………………………………………………….....IV
目錄 ..…………………………………………………………………VII
圖目錄 ..………………………………………………………………..X
表目錄 .……………………………………………………………...XIX
第一章 緒論 1
1-1 前言 1
1-2 研究目的 2
第二章 文獻探討 4
2-1 微藻 4
2-1-1 微藻概述 4
2-1-2 微藻培養 5
2-1-3 微藻培養基質滅菌方式 6
2-2 微波 10
2-2-1 微波作用原理 10
2-2-2 微波應用 12
第三章 實驗材料與方法 14
3-1 實驗設計 14
3-2 實驗材料與設備 16
3-2-1 微藻藻種 16
3-2-2 試驗菌株 16
3-2-3 海水培養基 17
3-2-4 固態培養基 17
3-2-5 細胞密度與吸光值量測方法 18
3-2-6 葉綠素螢光量測 18
3-2-7 連續式微波滅菌可行性試驗微波設備 19
3-2-8 連續式單模共振微波系統之滅菌藻水、菌液與海水試驗微波設備 22
3-3 實驗方法 27
3-3-1 連續式微波滅菌可行性試驗方法 27
3-3-2 連續式單模共振微波系統之滅菌藻水試驗方法 28
3-3-3 連續式單模共振微波系統之滅菌菌液試驗方法 30
3-3-4 連續式單模共振微波系統之海水滅菌試驗方法 32
3-3-5 平均加熱功率計算 33
第四章 結果與討論 34
4-1 連續式微波滅菌可行性試驗 34
4-2 連續式單模共振微波系統之滅菌藻水試驗結果 43
4-2-1 固態培養基培養微藻與雜菌之結果 47
4-2-2 離心管於恆溫震盪培養箱培養結果 57
4-2-3 微波腔體出水口溫度量測與平均加熱功率計算結果 ………………………………………………………...96
4-3 連續式單模共振微波系統之滅菌菌液試驗結果 99
4-3-1 固態菌落培養基試驗結果 100
4-3-2 微波腔體出水口溫度量測與平均加熱功率計算結果 ……………………………………………………….106
4-4 連續式單模共振微波系統之海水滅菌試驗結果 108
4-4-1 角毛藻細胞密度與吸光值量測結果 109
第五章 結論與建議 114
5-1 結論 115
5-2 建議 119
第六章 參考文獻 120


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