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研究生:高敏智
研究生(外文):Min Chih Kao
論文名稱:利用新型生化反應器探討澱粉生產酒精之最適化三菌共培養政策
論文名稱(外文):Studies on optimal co-cultural policies for ethanol production from starch using a novel bioreactor
指導教授:劉裕國
指導教授(外文):Y. K. Liu
學位類別:碩士
校院名稱:長庚大學
系所名稱:化工與材料工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:80
中文關鍵詞:生質能源澱粉共固定
外文關鍵詞:bioenergystarchco-immobilized
相關次數:
  • 被引用被引用:1
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摘要

本研究目的是探討以生物反應器作為發酵工具,並使用兩種澱粉分解菌Aspergillius awamori & Rhizopus japonicus共固定化培養,作為基質原料的馬鈴薯澱粉(Potato starch)經過固定於PU載體上的菌體分解形成葡萄糖後,再經由乙醇生產菌Zymomonas mobilis醱酵轉化為酒精,以作為再生能源使用之方法。

共固定系統包含固定於PU載體的兩種好氧菌及包埋於海藻酸鈣膠體中的Zymomonas mobilis。此三菌共固定於在單一反應器中進行共培養,以12 % (w/v.)進料濃度可得到較高的乙醇產量,在重複進料生產乙醇方面,分別以進料澱粉濃度6 % (w/v)、12 % (w/v)進行5批次重複培養,與單一批次發酵結果比較,雖然延長了培養時間但確實可得到較高乙醇產量,總乙醇產量分別是132.16g/L及241.54g/L。此外,在提高進料濃度之條件下,濃度24 % (w/v)之澱粉進料,因過高濃度之進料,導致乙醇的生產被抑制,為增加乙醇生產量並降低高濃度基質效應,在超過12 % (w/v)進料濃度時,採用饋料批次的培養政策可有效地提升乙醇的產量。

由結果可看出,此研究成果不僅可提高乙醇產量,對於生產效率的提升和設備的減少,更具有優勢。
Abstract

The aim of this study was to produce ethanol from potato starch by co-immobilized Aspergillus awamori, Rhizopus japonicus and Zymomonas mobilis using a novel bioreactor for the development of the renewable energy.

The co-immobilized system(A-R-Z system) contain two Starch decomposing mold, Rhizopus japonicus, Aspergillus awamori immobilized on PU carrier and one ethanol producing bacteria, Zymomonas mobilis immobilized on sodium alginate in a single reactor. The co-immobilized cells produced a high ethanol yield from 120 g/L potato starch. In repeated-batch fermentation process (five cycles) study, higher ethanol yield of 132.16 g/L and 241.54 g/L were achieved from 60 g/L potato starch and 120 g/L potato starch respectively, though time elongated. Higher starch concentration (240 g/L) feeding did not increase the ethanol yield. This ethanol production inhibition might be caused by high concentration substrate in the reactor. Further study showed step-feeding of soluble starch cultures could overcome this substrate inhibition and increase ethanol production.

The result proved the performance not only can enhance the ethanol yield, regarding production efficiency's promotion and equipment's reduction, has the superiority.
目錄

指導教授推薦書………………………………………………………

口試委員審定書………………………………………………………

國家圖書館授權書……………………………………………………iii

長庚大學授權書………………………………………………………iv

誌謝……………………………………………………………………v

中文摘要………………………………………………………………vi

英文摘要………………………………………………………………vii

目錄……………………………………………………………………viii

圖目錄…………………………………………………………………xii

表目錄…………………………………………………………………xv

第一章 緒論…………………………………………………………1

1.1前言………………………………………………………………1

1.2研究動機與目的…………………………………………………3

第二章 文獻回顧……………………………………………………6

2.1酒精汽油燃料之發展……………………………………………6

2.2酒精發酵菌 Zymomonas mobilis……………………………6

2.3糖化黴菌………………………………………………………8

2.4澱粉……………………………………………………………8

2.5澱粉醣化酶的種類和功能……………………………………9

2.5.1澱粉液化酶(α-Amylase) …………………………………10

2.5.2澱粉醣化酶(β-Amylase) …………………………………10

2.5.3葡萄糖澱粉分解酶(Glucoamylase) ………………………11

2.5.4葡萄糖苷酶(Glucosidase) …………………………………12

2.6澱粉分解酶之特性………………………………………………12

2.6.1澱粉分解酶之產物反饋抑制作用 …………………………12

2.6.2澱粉分解酶的誘導…………………………………………13

2.6.3熱穩定性……………………………………………………13

2.7微生物之培養政策……………………………………………13

2.7.1一般培養政策 ………………………………………………13

2.7.2共培養政策…………………………………………………15

2.8微生物的固定化………………………………………………16

2.8.1固定化的意義與優勢.………………………………………16

2.8.2固定化(immobilization)的方法……………………………17

2.8.3微生物之共固定化……………………………………20

2.9生物反應器……………………………………………………22

2.10實驗流程規劃………………………………………………23

第三章 材料與方法………………………………………………………25

3.1 菌株來源…………………………………………………………25

3.2 設備與藥品………………………………………………………25

3.2.1 設備儀器…………………………………………………….25

3.2.2 實驗藥品…………………………………………………26

3.2.3 培養基的配製.………………………………………………27

3.2.3.1 PDA培養基………………………………………… 27

3.2.3.2 預培養液態培養基……………………………………27

3.2.3.3 糖化醱酵液態培養基……………………………28

3.2.3.4 Zymomonas mobilis培養基…………………………29

3.2.4 試劑…………………………………………………………29

3.2.4.1 葡萄糖水劑……………………………………………29

3.3 實驗方法………………………………………………………30

3.3.1 菌種預培養…………………………………………………30

3.3.2 菌種的保存…………………………………………………30

3.3.3 分析方法……………………………………………………31

3.3.3.1 葡萄糖與乙醇測定……………………………………31

3.3.3.2 葡萄糖測定……………………………………………31

3.3.3.3 乙醇濃度測定…………………………………………32

3.3.4 澱粉轉化乙醇之理論產量…………………………………33

3.3.5 澱粉分解菌之共培養體系…………………………………33

3.3.6 凝膠包埋法之測試…………………………………………33

3.3.7 PU載體之製備 …………………………………………35

3.3.8 三菌共培養之糖化分解與乙醇發酵之探討……………35
.
3.3.9 饋料批次發酵之探討……………………………………36

第四章 結果與討論……………………………………………………39

4.1 A-R/Z單一反應器固定化共培養效應…………………………39

4.2 進料濃度在反應器培養中之效應……………………………41

4.3 重複進料在反應器培養中之效應……………………………43

4.4 高濃度澱粉重複進料在反應器培養中之效應………………44

4.5 饋料批次進料在反應器培養中之效應………………………46

4.6反應器乾燥澱粉進料培養中之效應…………………………49

第五章 結論與建議……………………………………………………53

5.1 結論 ……………………………………………………………53

5.2 建議 ……………………………………………………………54

參考文獻………………………………………………………………56

附錄 標準曲線……………………………………………………… 63
圖目錄

圖1 實驗計畫…………………………………………………………5

圖2 恩-杜氏代謝途徑…………………………………………………7

圖3澱粉結構…………………………………………………………9

圖4常見澱粉分解酶之切割位置………………………………………11

圖5 Glucosidase之分解形式…………………………………………12

圖6各種固定化方法……………………………………19

圖7膠體粒子的共固定化培養………………………………………21

圖8發泡載體的共固定化培養…………………………………………22

圖9系統開發與流程規劃…………………………………………24

圖10凝膠包埋法製作簡圖…………………………………34
.
圖11生物反應器構造圖………………………………37

圖12 A. awamori與R. japonicus於PU共固定培養一天之側視圖…38

圖13 A. awamori與R. japonicus於PU共固定培養二天之側視圖…38

圖14 A. awamori與R. japonicus於PU共固定培養三天之側視圖…38

圖15(A) A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)
及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中進行共培養之葡萄糖及乙醇變化…………………………40

圖15(B) A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)
及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中進行共培養之葡萄糖及乙醇變化(菌體重新活化)……41

圖16 以12%(w/v)澱粉進料,A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中進行共培養之葡萄糖及乙醇變化…………42

圖17以24%(w/v)澱粉進料,A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中進行共培養之葡萄糖及乙醇變化……………43

圖18以6%(w/v)澱粉進料,A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中,進行批次進料五次共培養之葡萄糖及乙醇變化………………43

圖19以12%(w/v)澱粉進料,A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中,進行批次進料五次共培養之葡萄糖及乙醇變化………………46

圖20以6%(w/v)澱粉進料,A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中,進行饋料批次共培養之葡萄糖及乙醇變化…………………………………………………………………48

圖21以24%(w/v)澱粉進料,A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中,進行饋料批次共培養之葡萄糖及乙醇變化…………………………………………………………………48

圖22以6%(w/v)乾燥澱粉進料,A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中,進行批次共培養之葡萄糖及乙醇變化…………………………………………………………………50

圖23以2%(w/v)乾燥澱粉進料,A. awamori與R. japonicus共固定於兩個橢圓形HR-08(上層)及HR-13(下層)之複合PU載體,加入Z. mobilis膠體於反應器中,進行饋料批次共培養之葡萄糖及乙醇變化…………………………………………………………………51





































表目錄

表1 各種固定化方法之優缺點比較表……………………………… 20

表2 PDA medium………………………………………………………27

表3 A. awamori與R. japonicus預培養液態培養基…………………28

表4 A. awamori與R. japonicus糖化醱酵液態培養基………………28

表5 Zymomonas mobilis培養基……………………………………… 29

表6 葡萄糖水劑成分………………………………………………… 30

表7 單一批次不同濃度澱粉之最高乙醇濃度及產量比較表.............45

表8 各培養政策生產乙醇之結果彙整表………………………….…52
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