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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/34601

Title: CBM複合益生菌冷凍乾燥技術研究及其應用於高濃度汙水處理
The Study of Lyophilization of Complex Beneficial Microorganisms (CBM) and Application of CBM in High Concentration Wastewater Treatment
Authors: Shih-Yun Lin
林詩紜
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: CBM複合益生菌;冷凍乾燥
Complex Beneficial Microorganisms;Lyophilization
Date: 2013
Issue Date: 2013-10-07T02:42:58Z
Abstract: 益生菌是指對宿主有益的微生物和其產物,應用在水產養殖上,直接影響產量、物質循環、動物營養、水質、疾病控制。過去在水產養殖上大都以光合菌與硝化菌作為水質與底質改善劑,但效果有限。本研究之目標為將複合益生菌應用在水產養殖上,為達到此一目標,本論文進行下列研究;(1)以即時定量聚合酶連鎖反應偵測定量複合型益生菌組合中各類微生物的含量;(2)以不同的抗凍劑將 CBM (Complex Beneficial Microorganisms) 菌進行冷凍乾燥製作 CBM 冷凍乾燥菌粉;(3)應用複合型益生菌處理高濃度豬尿廢水改善水質;(4)以總體基因體學及次世代定序之方法分析直接投餵 CBM 菌及投餵市售飼料之石斑魚腸道菌相差異。由於CBM 菌是一複合益生菌配方,有必要知道特定菌株在共棲培養過程中各類菌數的變化,本研究利用 Real-time PCR 對 CBM 菌進行絕對分子定量,其中 7 大類複合益生菌在共棲培養階段菌量為 2.13 × 108 - 4.10 × 109 CFU‧mL-1; 第一次擴量培養中,菌量為 1.16 × 106 - 6.77 × 108 CFU‧mL-1 ; 第二次擴量培養後的菌量為 3.12 × 105 - 7.94 × 108 CFU‧mL-1。使用不同抗凍劑進行 CBM 冷凍乾燥菌粉的製備實驗中,凍乾菌粉培養五天後利用 Real-time PCR 進行絕對分子定量,CBM 複合菌中除光合菌偵測不到菌數外,其餘各株菌菌數皆可達 1.53 × 105 - 5.59 × 107 CFU‧mL-1。在模擬廢水中加入 CBM 冷凍乾燥菌粉 100 ppm實驗中,CBM 冷凍乾燥菌粉仍能降低廢水中氨氮及 COD 含量,證明冷凍乾燥後的菌份仍有活性。在水質處理試驗中,添加 CBM 菌可改善養豬廢水的氨氮、亞硝酸、COD 及總磷含量;利用額外添加填料吸附 CBM 菌之生物厭氧滲濾系統可有效降低廢水中的氨氮、亞硝酸、COD、總磷及懸浮固體含量。利用複合型生物處理系統可有效降低廢水中的氨氮、亞硝酸、COD、總磷及懸浮固體含量,並能夠達到河川放流水排放標準。石斑魚腸道總體基因體學經次世代定序後結果顯示,投餵 CBM 菌飼料組別病原菌菌數與投餵市售商業飼料組別相比有減少的趨勢。
Probiotics, which are micro-organisms or their products with health benefit to the host, which have found useful in aquaculture as a means of disease control, supplementing or even in some cases replacing the use of antibiotics. Water quality is the most important essentials in aquaculture industry. In past, it has been shown most probiotics can improve water quality, there are phototrophic bacteria and nitrifying bacteria. The objective of present study is using Complex Beneficial Microorganisms (CBM) in aquaculture. To accomplish this objective, several specific aims were established; 1) apply real-time quantitative polymerase chain reaction (RT-qPCR) to quantitate composite probiotic of CBM which contains various of strains of microorganisms, 2) use different types of antifreeze agent in the freeze-drying process to produce the CBM powder. 3) apply of CBM in simulated wastewater treatment for improving water quality. 4) use metagenomics and next generation sequencing (NGS) to analysis of the difference microflora in grouper intestinal between direct-fed-microbial CBM and commercial feed. CBM is a composite probiotic formula by symbiotic fermentation process. Therefore, strain-specific quantification is not easy. In present study, a Q-PCR absolute quantification method was applied to quantify the amount of each strain on CBM, containing 7 types of microorganisms. During symbiotic culture, the microbial number were ranging from 2.13 × 108 to 4.10 × 109 CFU‧mL-1 respectively. The cells number in the first molasses amplification culture process were ranging from 1.16 × 106 to 6.77 × 108 CFU‧mL-1 respectively. The cell numbers in second molasses amplification culture process were ranging 3.12 × 105 to 7.94 × 108 CFU‧mL-1 respectively. Quantitative bacterial content of the CBM powder by real-time PCR analysis, the microbial number were ranging from 1.53 × 105 to 5.59 × 107 CFU‧mL-1 respectively, in addiontion to photosynthetic bacteria. Then CBM powder can reduce the level of ammonia, and COD in wastewater. In water quality treatment tests, adding CBM into pig urine wastewater, had shown that the ammonia, nitrite, COD, and total phosphorus levels were reducing significantly. Using Biological Anaerobic Infiltration System which coating CBM can reduce more ammonia, nitrite, COD, total phosphorus, and suspended solids. Furthermore using Complex Biological Treatment System can reduce more ammonia, nitrite, COD, total phosphorus, and suspended solids significantly, and can reach the discharge standards of the river effluent. And the metagenomics of the intestinal bacteria on grouper (Epinephelus coioides) showed that the pathogens bacteria which feeding CBM bacteria is less than feeding commercial feed fourteen days.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0010033036
http://ntour.ntou.edu.tw/handle/987654321/34601
Appears in Collections:[水產養殖學系] 博碩士論文

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