探討培養溫度對Pseudomonas taetrolens發酵乳清生產乳糖酸的影響

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羅雨承(Yu-Cheng Lo) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

探討培養溫度對Pseudomonas taetrolens發酵乳清生產乳糖酸的影響

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摘要(中)

在現今的社會，乳糖酸是一種被大量應用在人類生活之中的有機酸，舉凡食品、化妝品、製藥、生醫領域皆可見到其身影。乳糖酸之所以被如此廣泛的應用在各種不同的領域，主要是因為其具有出色的生物相容性、生物可分解性和抗氧化特性。目前製造乳糖酸的方法大多是利用化學合成的方式去生產，但合成過程卻會使用到對環境具有汙染性的金屬催化劑。然而，近年來發現了一種利用微生物發酵的方式去產生乳糖酸的方法。此方法即是使用Pseudomonas taetrolens發酵乳清生產乳糖酸。乳清是乳品製造中伴隨產生的副產物，每年全球產生的乳清中大約有30%沒有被妥善的運用，可能作為動物飼料抑或是農業用肥料，甚至是直接丟棄。而利用微生物發酵乳清生產乳糖酸來取代化學合成法可以避免使用到對環境不友善的催化劑，且微生物發酵法是利用廢棄物產生高附加價值產物的方法，其成本相對低廉，對環境也較友善，符合永續經營的概念。
　　在微生物發酵中，環境溫度會大大地影響微生物的生理狀態和發酵行為，所以，此論文的目的便是探討在不同的培養溫度下對Pseudomonas taetrolens發酵乳清生產乳糖酸的影響，其中，討論了種菌培養時間、種菌培養溫度、搖瓶生產溫度、放大規模的生物反應器生產溫度及兩階段溫度操作，最後，在針對實驗結果做出產物動力學分析。由實驗結果發現，最佳的種菌培養時間為24小時，最佳種菌培養溫度為35℃，搖瓶最佳生產溫度為30℃，在此溫度乳糖酸產量及產率最高，但生產溫度為20℃時所獲得的菌重最多，因此利用兩階段溫度操作，第一階段溫度設定20℃促使產生較多菌重，然後第二階段再調整至30℃以利產物生成，在此操作下乳糖酸的產量為29.793 g/L；產率為0.198 g/L h，比起固定生產溫度30℃的操作產量提升了約14%；產率部分則提升了約13%，在生物反應器規模的生產溫度實驗，得到了類似搖瓶規模的實驗結果，不同的是兩階段溫度操作時，產率可比固定生產溫度30℃提升約20%，且明顯縮短了發酵時間。根據產物動力學分析，可以看出較佳的發酵表現中，其產物皆較屬於混合生長相關型，因此，若調整發酵溫度將可促使產物生成趨近理想的動力學模式，進而改善與加強發酵表現。

摘要(英)

In today′s society, lactobionic acid is a kind of organic acid which is widely used in human life, it finds extensive applications in the fields of food, cosmetics, pharmaceuticals and biomedicine. It could be used in so many different fields, mainly because of its excellent biocompatible, biodegradable and antioxidant properties. Most of the current methods of producing lactobionic acid are produced by chemical synthesis, but the synthesis processes use harmful metal catalysts. However, in recent years, a method for producing lactobionic acid has been discovered by microbial fermentation. This method is producing lactobionic acid from whey by Pseudomonas taetrolens. Whey is the by-product produced in the manufacture of dairy products. About 30% of whey remains underutilized, most ending up being employed as animal feed, spread on agricultural land as fertilizer or even as waste. Producing lactobionic acid from whey by microorganism instead of using the chemical synthesis method can avoid the use of harmful catalysts. Furthermore, the microbial fermentation produces high value-added products from inexpensive feedstock such as polluting waste, so the cost of production is relatively low, and is more environmental friendly. It is consistent with the concept of sustainable development.
In microbial fermentation procedures, environmental temperature will greatly affect not only the physiological status but also the behavior of microorganisms. Ergo, the aim of this study was to investigate the effects of seed culture temperature and fermentation temperature on the production of lactobionic acid from whey by Pseudomonas taetrolens. Hence, the seed culture age, seed culture temperature, fermentation temperature in shake-flask and bioreactor, and two-stage fermentation temperature operation were all discussed in the present study. Finally, illustrate the ideal fermentation mode via the analysis of the product formation kinetics. The results of experiments showed that the optimal seed culture temperature was 35℃, and then the optimal fermentation temperature in shake-flask and bioreactor was 30℃. In this optimal operation, the yield and productivity of lactobionic acid were the highest among other operations, but the highest biomass concentration was obtained from the operation of which fermentation temperature was set at 20℃. Therefore, using the two-stage fermentation temperature operation, the first stage fermentation temperature was set at 20℃ to cause more biomass, and then the second stage fermentation temperature was shifted to 30℃ to enhance the production of lactobionic acid. In this operation in shake-flask, the yield of lactobionic acid was 29.793 g/L, which was about fourteen percent higher than that obtained from the operation of which fermentation temperature was fixed at 30℃. Besides, the productivity of lactobionic acid was 0.198 g/L h, which was about thirteen percent higher than that obtained from the operation of which fermentation temperature was fixed at 30℃. The results of fermentation temperature experiment conducted in bioreactor showed that the two-stage fermentation temperature operation shortened the fermentation time and the productivity of lactobionic acid was about twenty percent higher than that obtained from the operation of which fermentation temperature was fixed at 30℃. According to the analysis of the product formation kinetics, if the product formation is mixed-growth associated type, there will be a better fermentation performance. As a result, adjusting the seed culture temperature or fermentation temperature can make the product formation approaching the ideal type of the product formation kinetics model, thereby improving and enhancing the fermentation performance.

關鍵字(中)

★ Pseudomonas taetrolens
★ 溫度
★ 乳清
★ 乳糖酸
★ 產物動力學

關鍵字(英)

論文目次

摘要....................................................i
ABSTRACT...............................................ii
致謝...................................................iv
目錄....................................................v
圖目錄................................................viii
表目錄..................................................xi
一、緒論.................................................1
1-1　　研究背景..........................................1
1-2　　研究目的..........................................3
二、文獻回顧.............................................4
2-1　　乳糖酸的介紹......................................4
2-1-1　乳糖酸的基本性質...................................4
2-1-2　市場價值..........................................5
2-1-3　製造方法..........................................7
2-2　　實驗菌株的介紹....................................8
2-3　　乳清的介紹........................................9
2-4　　影響發酵表現的物理化學因子.........................10
2-4-1　培養基組成.......................................10
2-4-2　接菌量...........................................11
2-4-3　培養時間.........................................12
2-4-4　酸鹼值...........................................12
2-4-5　通氣速率.........................................12
2-4-6　攪拌速率.........................................12
2-4-7　發酵方式.........................................13
2-4-8　光照.............................................13
2-4-9　溫度.............................................13
三、實驗材料與方法.......................................14
3-1　　實驗流程.........................................14
3-2　　實驗規劃.........................................15
3-3　　實驗材料.........................................16
3-3-1　實驗菌種.........................................16
3-3-2　實驗藥品.........................................16
3-3-3　實驗儀器與設備....................................17
3-4　　實驗方法.........................................18
3-4-1　菌種固態培養.....................................18
3-4-2　種菌培養時間實驗..................................18
3-4-3　種菌培養溫度實驗..................................20
3-4-4　搖瓶規模之乳糖酸生產溫度實驗.......................21
3-4-5　搖瓶規模之乳糖酸兩階段生產溫度實驗..................23
3-4-6　生物反應器規模之乳糖酸生產溫度實驗..................24
3-4-7　生物反應器規模之乳糖酸兩階段生產溫度實驗............26
3-5　　分析方法.........................................28
3-5-1　pH值分析.........................................28
3-5-2　菌重濃度分析......................................28
3-5-3　還原糖濃度分析....................................29
3-5-4　乳糖酸濃度分析....................................31
3-5-5　產物動力學分析....................................33
四、結果與討論...........................................35
4-1　　不同的種菌培養時間對發酵影響.......................35
4-2　　不同的種菌培養溫度對發酵影響.......................38
4-3　　搖瓶規模下不同的乳糖酸生產溫度對發酵影響............41
4-4　　搖瓶規模之乳糖酸兩階段生產溫度實驗結果..............44
4-5　　生物反應器規模下不同的乳糖酸生產溫度對發酵影響.......47
4-6　　生物反應器規模之乳糖酸兩階段生產溫度實驗結果.........50
4-7　　產物動力學之討論..................................52
4-7-1　種菌培養溫度實驗之產物動力學分析...................52
4-7-2　搖瓶規模乳糖酸生產溫度實驗之產物動力學分析..........56
4-7-3　生物反應器規模乳糖酸生產溫度實驗之產物動力學分析.....60
五、結論與建議...........................................64
5-1　　結論.............................................64
5-2　　建議.............................................65
參考文獻................................................67

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［52］ Robert Luedeking and Edgar L. Piret “A kinetic study of the lactic acid fermentation. Batch process at controlled pH”, Biotechnology and Bioengineering, Vol. 1, Iss. 4, pp. 393-412, December 1959.

指導教授

徐敬衡(Chin-Hang Shu)

審核日期

2017-8-14

推文