| Summary: | 碩士 === 國立中興大學 === 生物產業機電工程學系所 === 103 === 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.
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