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研究生:歐姆
研究生(外文):J.Om Murugan
論文名稱:利用本土海洋弧菌Vibriospp.M20生產Poly(3-hydroxybutyrate)之培養最適化
論文名稱(外文):Media optimization of poly(3-hydroxybutyrate) from the indigenous marine isolateVibrio spp. M20
指導教授:魏毓宏
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:71
中文關鍵詞:Vibrio spp. M20poly(3-hydroxybutyrate)無機鹽
外文關鍵詞:Vibrio spp. M20poly(3-hydroxybutyrate)mineral salts
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一個分離自海底沉積物的海洋細菌Vibrio spp. M20,在本研究中用來評估生產生物可分解性塑膠poly(3-hydroxybutyrate,P(3HB))。本研究主要針Vibrio spp. M20量產P(3HB),開發一最適化培養基組成與配方。研究顯示:當以甘油做為主要碳源時,P(3HB)最大含量大約可達50%。研究近一步證實:加入NaOH、NH4OH以及 NaOCl等有利於P(3HB)之純化。此外,利用NaOCl分解非PHB之細胞其他物質時,P(3HB)之含量可達38% (MGYT);在使用NH4OH時P(3HB)之含量可達31% (MGYTT),而最佳的萃取條件為反應時間40分鐘並且在50℃情況下。在pH值降低時,會促進P(3HB)裂解酶分解細胞內之P(3HB),這種現象經推測,是由於加入某些無機鹽如檸檬酸鈉緩衝溶液(sodium citrate buffer)使pH值降至5.5造成P(3HB)含量下降。研究顯示:分別在MGYT中自12.3 % 降至8.2 %,而在MGYTT則自24% 降至 6%,幾乎有四倍的差異。本研究亦顯示:利用不同碳源、氮源以及氯化鈉(sodium chloride)濃度,明顯促進P(3HB)的累積,但添加無機鹽,將促使P(3HB)含量降低。
Vibrio spp. M20 a marine bacterium isolated from marine sediment was used to produce biodegradable polymer poly(3-hydroxybutyrate, P(3HB)). In order to enhance P(3HB) production, fascinating investigations including medium optimization, growth criteria under various conditions and fermentative strategies were carried out. A maximum P(3HB) content of about 50% were obtained when using glycerol as the sole carbon source. Gradually the extraction chemicals such as NaOH, NH4OH and NaOCl showed efficient digestion of non PHB cellular materials (NPCM). Also digestion with NaOCl results in 38% P(3HB) in MGYT media and NH4OH results in 31% (P3HB) in MGYTT media, while considering the digestion time length 40 min at 50℃ showed the best fit for extraction. Interestingly when the pH gets reduced it induces the depolymerase enzyme which results in intracellular P(3HB) degradation, this phenomenon has been speculated from adding mineral salts individually, followed by using the sodium citrate buffer at low pH 5.5 resulting in degradation of P(3HB) which leads to decrease of P(3HB) content from 12.3 % to 8.2 % in MGYT and 24% to 6% in MGYTT nearly 4 fold times. This experimental study also shows efficient carbon source, nitrogen source and sodium chloride concentration which facilitates P(3HB) accumulation. Simultaneously effect of individual addition of mineral salts on degradation of P(3HB) content has also been analyzed.
Content
Abstract (Chinese version) I
Abstract (English version) III
Acknowledgement V
Table of Contents VII
List of Tables X
List of Figures XII
Chapter 1 Introduction 1
1-1 Background of the study………………………………………………… ..1
1-2 Biodegradable polymers…………………………………………………. 1
1-3 Polyhydroxybutytrate–overview………………………………………… 4
1-4 Metabolic pathway for the synthesis of PHB……………………………. 8
1-5 Properties of PHB………………………………………………………. 10
1-6 Practical applications of PHB…………………………………………. ... 11
1-7 Objective of the study…………………………………………………... 12

Chapter 2 Materials and Methods…………………………………..............13
2-1 Materials..................................................................................................13
2-1-1 List of laboratory instruments …….……. … ..…….. 13
2-1-1 List of chemicals …14
2-2 Methods...................................................................................................16
2-2-1 Bacterial Strain 16
2-2-2 Culture storage condition 16
2-2-3 Types of culture medium used 16
2-2-4 Inoculums development 18
2-2-5 Optimization of Medium......................................................................18
2-2-5-1 Investigation of carbon source......................................................... .18
2-2-5-2 Investigation of nitrogen source........................................................18
2-2-5-2 Investigation of complex nitrogen source.........................................18
2-2-5-3 Investigation of sodium salt concentration........................................19
2-2-6 Analysis of Temperature on P(3HB) production...................................19
2-2-7 Analysis of inoculum level on P(3HB) production............................... 19
2-2-8 Analysis of agitation rate on P(3HB) production.................................. 19
2-2-9 Analysis of pH on P(3HB) production................................................. 20
2-2-10 Recovery of P(3HB) by simple digestion with chemicals ........... . ... 20
2-2-11 Influence of mineral salts……………………………………………20
2-2-12 Analysis of (P3HB) content from cell pelletd using sodium citrate buffer.… ...…………….. 22
2-2-13 Determining the cell concentration ..23
2-2-14 Cell dry weight 23
2-2-15 Gas chromatographic analysis .25

Chapter 3 Results and Discussions………………………………….............26
3-1 Effect of carbon source............................................................................. 26
3-2 Effect of nitrogen source.......................................................................... 28
3-3 Effect of complex nitrogen source............................................................30
3-4 Effect of sodium salt concentration.......................................................... 32
3-5 Effect of Temperature on P(3HB) production.......................................... 34
3-6 Effect of various inoculum level on P(3HB) production..........................36
3-7 Effect of agitation rate on P(3HB) production..........................................37
3-8 Effect of pH on P(3HB) production..........................................................39
3-9 Feeding strategy on P(3HB) production................................................... 42
3-10 Effective recovery of P(3HB) by simple digestion with chemicals…... 44
3-10-1 Effect of digestion temperature and digestion time length................. 47
3-11 Effect of mineral salts on P(3HB) production........................................ 52
3-12 Analysis of P(3HB) content from cell pellets treated with sodium citrate buffer....................................................................................................... 57
3-13 Analyzing the presence of depolymerase enzyme.................................. 59
3-14 Vibrio spp.M20 in 3L Fermenter............................................................ 62
3-15 GC result interpretation for P(3HB) obtained from GYT, MGYT and MGYTT media....................................................................................... .64
Chapter 4 Conclusions……………………………………………………… .66
References……………………………………………………68

List of Tables
Table 1-1 Effect of substrate cost and PHB yield on the
production cost of PHB………………………………………….. 6
Table 1-2 Properties of PHB………………………………………………...10
Table 2-1 Media composition for GYT…………………………………….. 17
Table 2-2 Media composition for MGYT…………………………………... 17
Table 2-3 Media composition for MGYTT………………………………… 17
Table 2-4 Fermenter condition……………………………………………… 23
Table 3-1 Analysis of P(3HB) content, concentration and CDW by MGYT on three different temperature……………………………………. 35
Table 3-2 Analysis of P(3HB) content, concentration and CDW by MGYTT on three different temperature………………………………… 35
Table 3-3 Investigating the effect of inoculum level in MGYT media…….. 36
Table 3-4 Investigating the effect of inoculum level in MGYT media……. ..36
Table 3-5 CDW, P(3HB) content and P(3HB) concentration of Vibrio spp. M20 in various concentration of carbon and nitrogen source (Fed Batch)-feeding regime analysis……………………….....……..43
Table 3-6 Digestion using various chemicals in cells obtained from MGYTT media……………………………………………………… .…..45
Table 3-7 Digestion using various chemicals in cells obtained from MGYT media…………………………………………...………………46
Table 3-8(a) Effect of digestion temp on recovery of P(3HB) and digestions were carried out for 1 h in cells obtained from MGYTmedia….47
Table 3-8(b) Effect of digestion temp on recovery of P(3HB) and digestions were carried out for 1 h in cells obtained from MGYTT media …………………………………………………….….....48
Table 3-9(a) Time profile of the purity of recovered P(3HB) and digestion reactions were carried out at 50℃ for 40 min………………..49
Table 3-9(b) Time profile of the purity of recovered P(3HB) and digestion reactions were carried out at 50℃ for 80 min………………..50
Table 3-9(c) Time profile of the purity of recovered P(3HB) and digestion reactions were carried out at 50℃ for 120 min……………….51

List of Figures
Figure 1-1 General structure of PHAs…………………………………......3
Figure 1-2 PHA Evolution…………………………………………………7
Figure 1-3 Metabolic pathway for PHB……………………….…………..9
Figure 2-1 Schematic diagram of Fermenter………………….…….……24
Figure 3-1 Vibrio spp. M20 in various carbon source (a) optical density and
(b) pH……………………………..…….…………….……....27
Figure 3-2 Effect of various carbon sources on synthesis of P(3HB)….....27
Figure 3-3 Optical density of Vibrio spp. M20 in various concentration of nitrogen source……………………………….……...….….....29
Figure 3-4 Effect of various concentration of Yeast Extract on P(3HB) and
CDW………………………………….……….……….…......29
Figure 3-5 Optical density of Vibrio spp. M20 in various concentration of complex nitrogen source……………………….……………..31
Figure 3-6 Effect of various concentration of complex nitrogen source in synthesis of P(3HB)………………………..............................31
Figure 3-7Vibrio spp.M20 in various concentration of NaCl (a) optical density
(b) pH……….……………………………………………....... 33
Figure 3-8 Effect of various concentration of NaCl in P(3HB) synthesis……………………………………………….….......33
Figure 3-9 Effects of agitation rate on P(3HB) production in (a) GYT media, (b) MGYT media and (c) MGYTT media………..…………38
Figure 3-10 Vibrio spp. M20 in MGYT media under various pH
(a) optical density (b) pH…………………………..………..40

Figure 3-11 Effect of MGYT media under various pH in synthesis of P(3HB)……………………………………….………….…..41
Figure 3-12 Vibrio spp. M20 in MGYTT media under various pH (a) optical density (b) pH.…………………………………......................41
Figure 3-13 Effect of MGYTT media under various pH in synthesis of P(3HB)……………………………………………………….41
Figure 3-14 Vibrio spp. M20 in different concentration of carbon and nitrogen source (Fed Batch)-Feeding Regime analysis (a) optical density and (b) pH..……….………………………...…......................43
Figure 3-15 Vibrio spp. M20 in mineral salts individually adding (a) optical density and (b) pH…….…..………………………… .….….53
Figure 3-16 Effect of various mineral salts (by adding) in synthesis of P(3HB).…………………………………………...................53
Figure 3-17 Vibrio spp. M20 in mineral salts individually ignoring (a) optical density and (b) pH…………..………………………………54
Figure 3-18 Effect of various mineral salts (by ignoring) in synthesis of P(3HB)…………………………………………………..…..54
Figure 3-19 Effect of pH adjustment on individual addition of mineral salts………………………………………………………......55
Figure 3-20 Analysis of pH adjustment during individual addition of mineral salts………………………………………………………….56
Figure 3-21 CDW, P(3HB) content and P(3HB) concentration in
sodium citrate buffer at various pH in MGYT media……….58
Figure 3-22 CDW, P(3HB) content and P(3HB) concentration in
sodium citrate buffer at various pH in MGYT media…..…...58

Figure 3-23 Effect of different pH on analysing the presence of depolymerase enzyme……………………………………………..…..…......60
Figure 3-24 Effect of different temperatures on analysing the presence of depolymerase enzyme………………………..…………….....60
Figure 3-25 Effect of different metal ions on analysing the presence of depolymerase enzyme……………………………..…..……...61
Figure 3-26 Time course profiles of CDW, Residual CDW, P(3HB) concentration and P(3HB) content in MGYT……...……..…..63
Figure 3-27 Time course profiles of CDW, Residual CDW, P(3HB) Concentration and P(3HB) content in MGYTT………......…..63
Figure 3-28 GC peak for P(3HB) (a) obtained from sigma, (b) obtained from GYT media, (c) obtained from MGYT media, (d) obtained from MGYTT media………………………………..……………....65
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