邱文英臺灣大學：化學工程學研究所何宜靜Ho, I-ChingI-ChingHo2007-11-262018-06-282007-11-262018-06-282006http://ntur.lib.ntu.edu.tw//handle/246246/52154本研究利用Pseudomonas oleovorans發酵生產生物可分解性聚酯PHA，調整培養條件以提高產量，並利用含不同官能基之碳源及不同方式進行培養，期能生產具不同結構之PHA以增加應用性。 以辛酸鈉為培養碳源進行培養條件的調整，探討因素包括碳氮源及無機鹽類濃度，實驗結果發現，磷酸氫二鉀或磷酸二氫鉀不足會抑制菌體生長及PHA累積，而高濃度硫酸鎂對菌體生長有幫助，但會降低PHA產量；同時考慮菌量及PHA累積下，磷酸氫二鉀、硫酸鎂及碳源最適濃度各為5.8 g/L、0.4 g/L及48 mM，而C/N ratio (w/w)在3~5間可累積最多之PHA。嘗試添加非單體相關碳源以降低生產成本，發現此類碳源僅利於菌體生長，其中以葡萄糖2 g/L及辛酸鈉6 g/L混合碳源培養可獲得較多菌量及PHA；綜合以上結果進行發酵槽培養，並利用pH變化做饋料指標且維持氮源濃度小於1 g/L，經葡萄糖及辛酸鈉混合碳源饋料一次，可得1.996 g/L之PHA。 欲得含苯環及雙鍵等官能基之PHA，P. oleovorans分別以苯戊酸、亞麻油酸、油酸及辛烯酸進行培養，所生合成之PHA各為含苯環結構之PHPV (poly-3-hydroxy-5-phenylvalerate)、含三種飽和鏈段單體/兩種不飽和鏈段單體之共聚物、含四種飽和碳鏈單體/一種具單一雙鍵單體之共聚物及不含雙鍵之HO (3-hydroxyoctanoate)/ HC (3- hydroxycaproate)共聚物。 添加兩種不同碳源並改變培養方式，以獲得含不同結構及形式之PHA。使用辛酸鈉及十一烯酸兩碳源進行培養下，當添加辛酸鈉及十一烯酸混合碳源主培養，產物組成不因PHA累積或分解而變，且Tg及Tm隨產物組成比例不同而變化，推論此PHA為一random copolymer；若使用辛酸鈉及限氮狀況進行主培養，培養16小時後饋入等濃度之十一烯酸，可得1.257 g/L之PHO/ PHU混掺物(blend)，產物含雙鍵比例達25 %。使用十一烯酸及苯戊酸兩碳源培養下，添加不同比例之十一烯酸及苯戊酸混合碳源，推測會生合成PHU/ PHPV混掺物，隨十一烯酸添加量增加，PHA累積量及產物中雙鍵含量均會提高。Biosynthesis of biodegradable PHA (polyhydroxyalkanoate) using Pseudomonas oleovorans was investigated. Improve production of PHAs by regulating the culture conditions. PHAs with various side chain and physical properties were expected from carbon sources with functional group. Using sodium octanoate as carbon source, effect of concentration of carbon source, nitrogen source, and inorganic salts was examined. Cell growth and PHA accumulation were unfavorable with insufficient K2HPO2 and KH2PO4. High concentration of MgSO4 promoted cell growth but reduced PHA content. Considering DCW (dry cell weight) and PHA content, the optimal concentration of K2HPO4, MgSO4, and carbon source were 5.8 g/L, 0.4 g/L, and 48 mM, respectively. And the highest weight of PHA was obtained when C/N ratio (w/w) ranged between 3 and 5. To reduce the cost, non-related carbon sources were used and the results showed that non-related carbon sources were simply advantageous to cell growth. Among them, highest DCW and weight of PHA were attained with 2 g/L of glucose and 6 g/L of sodium octanoate. Fermentor cultivation was conducted by combining the above optimal culture condition. Feeding mixed carbon source of glucose and sodium octanoate once by the change of pH, weight of PHA was 1.996 g/L harvested from fermentor. Four different carbon sources were dosed to introduce functional groups of PHAs in this work, and structure of every PHA was presented as follows. PHPV (poly-3-hydroxy-5-phenylvalerate) was produced when 5-phenylvaleric acid was dosed. PHA contained three species of saturated monomer and two species of monomer with double bond when linoleic acid was used as a sole carbon source. Copolymer with four species of saturated monomer and one unsaturated monomer was biosynthesized from oleic acid. Dosing 2-octenoic acid, the PHO-co-HC (poly-3- hydroxy-octanoate-co-3-hydroxycaproate) was obtained. Structure, polymer form－blend or copolymer, and physical property of PHA varied with carbon sources and fermentation method. A random copolymer of PHA was supposed when sodium octanoate and undecylenic acid were used as mixed carbon source. Composition of PHA was not changed with accumulation or degradation of PHA. In addition, Tg and Tm were different according to composition of PHA. When main fermentation of sodium octanoate with nitrogen limitation and then feed of undecylenic acid was conducted, 1.257 g/L of PHO/ PHU blend was obtained. The PHA grown with mixture of undecylenic acid and 5-phenylvaleric acid was a blend of PHU and PHPV. Weight of PHA and ratio of unsaturated unit increased with dosage of undecylenic acid.中文摘要...I Abstrac...III 目錄...V 圖目錄...IX 表目錄...XV 第一章 緒論...1 1-1 前言...1 1-2 研究目的...1 第二章 文獻回顧...3 2-1 生物可分解性高分子...3 2-2 PHA之簡介...4 2-3 PHA之發展簡史...5 2-4 利用細菌生產PHA之代謝過程...7 2-5 發酵生產PHA之探討...8 2-6 Pseudomonas oleovorans發酵生產PHA...10 2-7 不同側鏈結構PHA之簡介...15 2-8 使用兩種以上碳源培養之PHA...17 2-9 PHA之延伸研究...19 2-10 PHA之應用及目前市場狀況...21 第三章 材料與方法...23 3-1 實驗藥品與試劑...23 3-2 實驗儀器...25 3-3 實驗流程及方法...26 3-3.1 菌種來源...28 3-3.2 培養基組成...28 3-3.3 菌種活化與種菌培養...29 3-3.4 發酵槽操作流程...30 3-3.5 基質及產物之分析方法...31 3-3.5.1 銨氮濃度之測定...31 3-3.5.2 菌體乾重之測定...32 3-3.5.3 PHA之萃取與純化...32 3-3.5.4 核磁共振儀(NMR)分析...33 3-3.5.5 微差熱掃瞄分析儀(DSC)分析...33 3-3.5.6 凝膠滲透層析儀(GPC)分析...33 3-4 培養條件對Pseudomonas oleovorans發酵生產PHA之影響...34 3-4.1 初始pH值影響之探討...34 3-4.2 氮源及C/N ratio影響之探討...34 3-4.2.1 以硫酸銨及yeast extract為氮源之比較...34 3-4.2.2 不同碳氮源濃度與C/N ratio之影響...34 3-4.3 磷酸氫二鉀與磷酸二氫鉀濃度影響之探討...34 3-4.3.1 不同濃度磷酸氫二鉀與磷酸二氫鉀之影響...34 3-4.3.2 緩衝液型及非緩衝液型培養基之影響...35 3-4.3.3 磷酸氫二鉀濃度之影響...35 3-4.4 硫酸鎂濃度影響之探討...35 3-4.5 非單體相關碳源影響之探討...35 3-4.5.1 添加不同非單體相關碳源之影響...35 3-4.5.2 辛酸鈉與非單體相關碳源混合培養之影響...36 3-4.6 碳源濃度影響之探討...36 3-4.7 發酵槽培養實驗之探討...36 3-5 特殊碳源對菌體生長、PHA累積及PHA結構與物性之影響...37 3-5.1 以苯戊酸(5-Phenylvaleric acid, 5-PVA)為碳源發酵結果...37 3-5.2 以亞麻油酸(Linoleic acid)為碳源發酵結果...37 3-5.3 以油酸(Oleic acid)為碳源發酵結果...37 3-5.4 以辛烯酸(2-Octenoic acid)為碳源發酵結果...38 3-6不同混合碳源及培養方式對產物PHA結構與形式之影響...38 3-6.1 辛酸鈉及十一烯酸混合碳源之培養結果...38 3-6.2 以辛酸鈉為培養碳源且中途饋加十一烯酸之培養結果...38 3-6.3 純PHO及純PHU不同比例混掺物(blend)之製作...39 3-6.4 不同比例十一烯酸及苯戊酸混合碳源之培養結果...39 3-6.5 以十一烯酸為培養碳源且中途饋加苯戊酸之培養結果...39 3-6.6 純PHU及純PHPV不同比例混掺物(blend)之製作...40 第四章 結果與討論...41 4-1 培養條件對Pseudomonas oleovorans發酵生產PHA之影響...41 4-1.1 初始pH值影響之探討...41 4-1.2 氮源及C/N ratio影響之探討...43 4-1.2.1 以硫酸銨及yeast extract為氮源之比較...43 4-1.2.2 不同碳氮源濃度與C/N ratio之影響...43 4-1.3 磷酸氫二鉀與磷酸二氫鉀濃度影響之探討...48 4-1.3.1 不同濃度磷酸氫二鉀與磷酸二氫鉀之影響...48 4-1.3.2 緩衝液型及非緩衝液型培養基之影響...49 4-1.3.3 磷酸氫二鉀濃度之影響...49 4-1.4 硫酸鎂濃度影響之探討...53 4-1.5 非單體相關碳源影響之探討...54 4-1.5.1 添加不同非單體相關碳源之影響...54 4-1.5.2 辛酸鈉與非單體相關碳源混合培養之影響...54 4-1.6 碳源濃度影響之探討...60 4-1.7 發酵槽培養實驗之探討...62 4-1.7.1 以辛酸鈉為碳源發酵培養之結果...62 4-1.7.2 以辛酸鈉為培養碳源並進行一次饋料之結果...63 4-1.7.3 培養時間對分子量影響之探討...65 4-2 特殊碳源對菌體生長、PHA累積及PHA結構與物性之影響...74 4-2.1 以苯戊酸(5-Phenylvaleric acid, 5-PVA)為碳源發酵結果...74 4-2.2 以亞麻油酸(Linoleic acid)為碳源發酵結果...78 4-2.3 以油酸(Oleic acid)為碳源發酵結果...83 4-2.4 以辛烯酸(2-Octenoic acid)為碳源發酵結果...88 4-3 不同混合碳源及培養方式對產物PHA結構與形式之影響...94 4-3.1 辛酸鈉及十一烯酸混合碳源之培養結果...94 4-3.2 以辛酸鈉為培養碳源且中途饋加十一烯酸之培養結果..109 4-3.3 不同比例十一烯酸及苯戊酸混合碳源之培養結果...116 4-3.4 以十一烯酸為培養碳源且中途饋加苯戊酸之培養結果..126 第五章 結論...130 參考文獻...132 附錄1 檸檬酸循環 (Tricarboxylic acid cycle；TCA cycle)...140 附錄2 銨氮濃度標準曲線...1411349397 bytesapplication/pdfen-US官能基聚羥基烷酯食油假單胞桿菌培養條件混合碳源functional grouppolyhydroxyalkanoatePseudomonas oleovoransculture conditionmixed carbon sourcethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52154/1/ntu-95-R93524012-1.pdf