Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22316
DC FieldValueLanguage
dc.contributor黃介辰zh_TW
dc.contributor吳禮字zh_TW
dc.contributor.advisor溫福賢zh_TW
dc.contributor.author陳建宇zh_TW
dc.contributor.authorChen, Chien-Yuen_US
dc.contributor.other中興大學zh_TW
dc.date2008zh_TW
dc.date.accessioned2014-06-06T07:17:43Z-
dc.date.available2014-06-06T07:17:43Z-
dc.identifierU0005-0802200721570300zh_TW
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Disruption of the sole ldhL gene in Lactobacillus sakei prevents the production of both L- and D-lactate. Microbiology 144 ( Pt 12):3327-33. 28. Marten, E. A., Sherrard, E. C., Peterson, W. H., Fred, E. B. . 1927. Production of Lactic Acid by Fermentation of Wood Sugar Remaining after Alcoholic Fermentation. Ind. Eng. Chem., Ind. Ed. 19:1162-1165. 29. Mason, C. K., M. A. Collins, and K. Thompson. 2005. Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool. J Microbiol Methods 60:353-63. 30. Messing, J. 1983. New M13 vectors for cloning. Methods Enzymol 101:20-78. 31. Miller, G. L. 1959. Use of dinitrosalicylic as reagent for the determination of reducing sugars. Anal. Chem. 31:426-428. 32. Nakasaki, K., and T. Adachi. 2003. Effects of intermittent addition of cellulase for production of L-lactic acid from wastewater sludge by simultaneous saccharification and fermentation. 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dc.identifier.urihttp://hdl.handle.net/11455/22316-
dc.description.abstract乳酸及其衍生物廣泛用於製藥、釀酒、食品、保健品、造紙、油漆、電鍍和塑料工業等。乳酸的光學異構物L-(+)-和D-(-)-乳酸經過聚合後可生成聚乳酸(polylactic acid, PLA),PLA是一種可再生利用、在自然情況下可被環境微生物降解的塑膠。藉由合成時控制L-(+)-和D-(-)-lactic acid 異構物的比例可以改變PLA的物理特性而賦予PLA製品各種不同的用途。目前乳酸的生產方式主要是利用乳酸菌將植物澱粉發酵,再將乳酸從發酵液中萃取純化出來,製造過程中原料和分離兩種異構物所需的成本是造成目前PLA價格居高不下的主要原因。若能利用有機廢棄物來生產乳酸,將可有效地降低 PLA 的生產成本,同時解決這些廢棄物的處置問題。Lactobacillus pentosus可兼行同型/異型乳酸醱酵,代謝六碳醣產生大量乳酸(D、L-form),或將五碳醣轉化成乳酸、醋酸與乙醇。有研究報告指出許多Clostridium 屬的細菌能夠形成胞外纖維分解酵素複合體(cellulosome),用來分解纖維素以獲得生長所需的碳源。但是Clostridium需要絕對厭氧的環境才能生長,而本實驗室有一株好氧菌Bacillus amyloliquefaciens I 可以用來營造絕對厭氧的環境,同時也具有分解纖維素等多種基質的能力。本研究嘗試利用這些菌株,探討以含有大量纖維素的民生下水污泥堆肥與造紙事業廢棄污泥作為生長基質,經由共培養發酵的方式來生產乳酸之可行性。實驗結果顯示民生下水污泥堆肥與造紙廠的廢水污泥經過L.pentosus批次培養發酵後,最高可分別產生5.02 g/L與2.69 g/L的乳酸。由於民生下水污泥堆肥來源不穩定且組成分過於複雜,本研究最後以造紙廠的廢水污泥作為營養基質進行生產乳酸的研究。將L. pentosus 和Clostridium M1 在添加營養鹽的廢水污泥稀釋液中進行共培養,34小時後乳酸生產達到最高,約為1.35 g/L,其中包括0.6 g/L的D-form乳酸、和0.76 g/L的L-form乳酸,但是在108小時之後只剩下約0.56 g/L的 L-form乳酸。L. pentosus 、 Clostridium M1 、Bacillus Ⅰ的共培養實驗最高可在48小時後產生約0.9 g/L的乳酸,但是在155小時之後也只剩下約0.83 g/L的L-乳酸。因此似乎經由這樣的方式即可獲得L-乳酸而不需再經過分離的步驟。培養時若給予適量的氮源,則L. pentosus、Clostridium M1與Bacillus I共培養的一組乳酸產量可在48小時到最高,為4.5 g/L,L. pentosus以及L. pentosus和Bacillus I共培養的兩組乳酸產量在48小時分別為4.8 g/L及4.6 g/L,均約為未添加氮源時的5倍。以上的結果顯示廢水污泥缺乏微生物生長所需的氮源,但若能適量外加富含氮源的廢棄物,例如造酒廠的廢酵母粉,將更能提升乳酸產量與基質利用率。由於作為基質的污泥並不需要任何的成本,加上乳酸具有較高的經濟效益,而發酵過的污泥則可作為垃圾掩埋場的覆土或土壤改良劑,所以可更進一步降低生產成本。 為了降低光學異構物分離的成本,本研究同時計畫以分子生物學的方式得到產生單一形式乳酸之L.pentosus 突變株。此部分實驗目前仍無結果還在進行檢討試驗中。zh_TW
dc.description.abstractLactic acid (LA) and its derivatives are versatile chemicals used in food, pharmaceutical, leather, and polylactic acid (PLA) plastics industries. Because of different optical rotation , lactic acid can be divided into L- lactic acid and D-lactic acid. Polylactic acid (PLA) could be synthesized by polyreaction of D- and L- lactic acid. PLA is a renewable , biodegradable plastic that can be degraded by environmental microorganisms. We can offer it different physical characteristics for specific application by altering the ratio of L- lactic acid and D-lactic acid isomers. Usually lactic acid is produced from the fermentation of starch by lactic acid bacteria , then followed by steps of extraction and purification. The cost of raw material and optical isomers isolation are the main reasons that cause the high price of PLA at present time. If lactic acid could be produced from organic waste material , the producing cost of PLA will be effectively reduced and the problems of handling these organic waste material will be solved simultaneously. Lactobacillus pentosus is a bacterium that can carry out either homo- or hetero-lactic acid fermentation, it produces large amount of lactic acid from hexoses, or produces lactic acid、ethanol and acetic acid from pentoses. In this study, L. pentosus was used to test the possibility of producing lactic acid from a regenerated resource, the wastewater sludge of paper mill. For growing as co-culture, a few Clostridium isolates, which can help to degraded cellulose in compost and sludge, and Bacillus amyloliquefaciens I, which can consume oxygen to make an anaerobic environment for lactobacilli and clostridia, were also inoculated into the culture. Experimental results indicated that the maximal lactic acid production was 2.69 g/L when L. pentosus was grown in a 10-ml batch culture with 0.2 g/ml sludge for 4 days. When L. pentosus was co-cultured with Clostridium M1, the production of lactic acid was increased to 1.35 g/L (including 0.6 g/L D-lactic acid and 0.76 g/L L-lactic acid) after a growth of 34 hours and existed only as L-(+) isomer (0.55 g/L) after 108 hours. When L. pentosus was co-cultured with Clostridium M1 and Bacillus Ⅰ, the production of lactic acid was 0.9 g/L (including 0.36 g/L D-lactic acid and 0.54 g/L L-lactic acid) after a growth of 48 hours and existed only as L-(+) isomer (0.83 g/L) after 155 hours. These results may be helpful to establish a L-lactic acid producing system without optical isomers purification. When nitrogen source supplemented to growth medium, the co-culture of L. pentosus 、Clostridium M1 and Bacillus Ⅰcould product 4.5 g/L of lactic acid after a growth of 48 hours, L. pentosus and the co-culture of L. pentosus and Bacillus Ⅰalso produced 4.8 g/L and 4.6 g/L of lactic acid, respectively, after a growth of 48 hours. All of these concentration of lactic acid were three times as much as those with out nitrogen source. These results also indicated that the wastewater sludge of paper mill lacks nitrogen source, therefore, perhaps organic waste material rich in nitrogen source could be used together with the wastewater sludge of paper mill to produce lactic acid. In order to produce pure L- lactic acid and D-lactic acid isomers separately, this study also tried to destroy the d-ldh and l-ldh genes of L. pentosus, respectively. This experiment is still in process at present.zh_TW
dc.description.tableofcontents中文摘要 i Abstract iii 第一章 前言 1 一、分解性塑膠 1 二、乳酸與聚乳酸(polylactic acid, PLA) 3 (1)乳酸的性質及用途 3 (2)聚乳酸(polylactic acid, PLA) 3 (3) PLA之應用 5 三、廢棄污泥問題 6 (1)目前廢棄污泥的處理方法 6 (2)台灣污水下水道發展 6 (3)廢棄污泥資源化的潛力 7 四、研究目的 8 第二章 材料方法 9 一、實驗材料 9 菌種與質體 9 藥品、酵素 11 培養基 11 試劑與緩衝溶液 12 表三、PCR及定序所使用之引子(primer) 14 二、實驗方法 15 乳酸菌之篩選 15 厭氧污泥培養基的配製方法 15 還原糖濃度測定 15 DNA之製備 16 1.、小量質體抽取 16 2、大量質體DNA的抽取 16 3、染色體DNA之抽取 17 聚合酶連鎖反應(polymerase chain reaction, PCR) 17 洋菜膠體電泳分析(agarose gel electrophoresis) 18 質體之構築與選殖(cloning) 18 1. 限制酶的切割(restriction enzyme digestion) 18 2. DNA回收 18 3. DNA的補齊(fill-in) 19 4. DNA的黏接反應(Ligation) 19 5、細胞轉形作用(transformation) 19 6. 快速質體篩選法 20 電穿孔法(Electroporation) 20 1.電穿孔勝任細胞之製備 20 2.電穿孔 20 乳酸產量測定 21 第三章 結果 22 一、利用廢棄污泥生產乳酸 22 (Ⅰ) L. pentosus基質利用特性分析 22 (Ⅱ) 污泥發酵特性分析 22 (1) L. pentosus利用污泥生長的情形與轉換污泥為乳酸的能力分析 22 (2) L. pentosus 將污泥醣化能力分析 23 (3) L. pentosus 分解污泥之能力分析 24 (Ⅲ)利用共培養方式提升乳酸產量之探討 24 (1) L. pentosus與Clostridium 的共培養實驗分析 25 (4)利用大型發酵槽(10 L)模擬乳酸生產 26 (5)污泥添加氮源共培養發酵 27 (6)最佳污泥濃度測試 27 二、運用分生技術生產單一形式乳酸 28 (Ⅰ) 構築基因突變所需之載體 28 (1)Single-crossover所需之載體 28 (2)Double-crossover所需之載體 28 (Ⅱ) 建立乳酸菌轉殖系統 29 三、乳酸菌篩選 29 第四章 討論 31 第五章 參考文獻 35zh_TW
dc.language.isoen_USzh_TW
dc.publisher生命科學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0802200721570300en_US
dc.subjectlactic aciden_US
dc.subject污泥zh_TW
dc.subjectLactobacillus pentosusen_US
dc.subjectsludgeen_US
dc.subjectco-cultureden_US
dc.subjectregenerated resourceen_US
dc.subjectldhen_US
dc.subject乳酸脫氫酶共培養zh_TW
dc.subject再生性資源zh_TW
dc.titleLactobacillus pentosus利用再生性資源生產乳酸之研究zh_TW
dc.titleProduction of lactic acid from regenerated resources by Lactobacillus pentosusen_US
dc.typeThesis and Dissertationzh_TW
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